B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum - starved in AβO - containing Hank ' s balanced salt solution for 2 hours . were then imaged for NiMA , after which insulin was immediately added , were then imaged again 30 minutes later . Note that NiMA occurred in absence or presence of AβOs , which inhibited NiMA in wild type human neuron Data information : Each colored lin refers to a single field of view containing 2 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Statistical analyses were performed using Student ' s two - unpaired t -

O O B-ORGANISM B-CELL_TYPE O B-GENEPROD O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-ORGANISM 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

B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin was immediately added, and the cells were then imaged again 30 minutes later. Note that NiMA occurred in the absence or presence of AβOs, which inhibited NiMA in wild type human neuron Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to ( R + L Each colored refers to a single field of view containing 5 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 , a downstream target of mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - unpaired t -

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

Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored refers to a single field of view containing 5 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 , a downstream target of mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - unpaired t -

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

Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


( D Human fibroblasts deficient in Tsc2 ( TSC2 Δexons1 - 14 ) were imaged before 30 minutes after insulin addition . Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or , were imaged before 30 minutes after stimulation . Each colored refers to a single field of view containing 2 - 5 , each pair of solid dotted lines of same color refers to same field of view Statistical analyses were performed using Student ' s two - unpaired t -

O O B-ORGANISM B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O O B-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

(D Human fibroblasts deficient in Tsc2 (TSC2 Δexons1-14) were imaged before and 30 minutes after insulin addition. Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student's two-tailed unpaired t-test


( E ) Re - expression of human Tsc2 rescues mitochondrial responses to insulin . Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or , were imaged before 30 minutes after stimulation . Each colored refers to a single field of view containing 2 - 5 , each pair of solid dotted lines of same color refers to same field of view . Re - expression of Flag - WT Tsc2 was corroborated by indirect immun o ( IF ) . Statistical analyses were performed using Student ' s two - unpaired t -

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

(E) Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immunofluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test


C Sequences of synthetic PrP alleles differing in octarepeat region of mousePrP . Conserved histidines in octarepeats 2 - 5 are coloured blue missense mutations are shown in red . Overproduction of C2 fragment is indicated by + + signs to right of panel .

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

C Sequences of synthetic PrP alleles differing in the octarepeat region of mousePrP. Conserved histidines in octarepeats 2-5 are coloured blue and missense mutations are shown in red. Overproduction of C2 fragment is indicated by ++ signs to the right of the panel.


A Lysates from RK13 transfected with WT , S1 , S3 S3 . F88W PrP were probed with antibody Sha31 , 9A2 12B2 after digestion with PNGaseF .

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A Lysates from RK13 cells transfected with WT, S1, S3 and S3.F88W PrP were probed with antibody Sha31, 9A2 and 12B2 after digestion with PNGaseF.


B , C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 lysates . A schematic of different lengths of C2 caused by single mutations in OR is shown in lower portion of ( C ) .

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B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).


D Lysates of N2a , HEK , SMB - PS SH - SY5Y not transfected ( − ) transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF analysed by Western blot using Sha31 .

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

D Lysates of N2a, HEK, SMB-PS and SH-SY5Y cells not transfected (−) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31.


E of lysates from RK13 transfected with plasmids incorporating Phe substitutions into hydrophobic domain of WT PrP , S1 PrP S3 PrP . Lysates were PNGaseF - digested before Western blot using Sha31 antibody . F1 : A114F , F2 : G118F , F3 : G122F , F4 :

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E Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP, S1 PrP and S3 PrP. Lysates were PNGaseF-digested before Western blot using the Sha31 antibody. F1: A114F, F2: G118F, F3: G122F, F4: G126F


A Brain homogenates of TgPrP ( WT ) , TgPrP ( S1 ) - 17 TgPrP ( S3 . F88W ) - 35 mice were analysed by Western blot before after PNGaseF digestion using PrP antibodies Sha31 , 12B2 9A2 . Prnp0 / 0 , WT brain homogenates were used as controls .

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A Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls.


B of C2 to full - PrP in TgPrP ( S3 . F88W ) - 35 WT mice ( left panel , n = 7 , P = 1 . 46E - 06 ) in RK13 expressing WT S3 PrP ( right panel , n = 5 , P = 4 . 01E - 05 ) is shown . Values are presented as mean ± . Unpaired , two - t - , * * * * P < 0 . 0001 .

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B The ratio of C2 to full-length PrP in TgPrP(S3.F88W)-35 and WT mice (left panel, n = 7, P = 1.46E-06) and in RK13 cells expressing WT and S3 PrP (right panel, n = 5, P = 4.01E-05) is shown. Values are presented as mean ± SEM. Unpaired, two-tailed t-test, ****P < 0.0001.


C Different tissue homogenates from a TgPrP ( S3 . F88W ) - 35 mouse were PNGaseF - digested analysed for PrP using antibody 1A6 . FL , full - PrP ; ns , non - specific signal detected in immunoblotting procedure .

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C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.


A Sections of sciatic nerve from TgPrP ( S3 . F88W ) - 14 mice ( 610 days old ) TgPrP ( S3 . F88W ) - 35 mice ( 614 , 570 614 days old , respectively ) were analysed using toluidine blue ( upper row ) , with third panels for each showing a hypermyelinated phenotype . Scale bar in left panel represents 50 μm . lower rows show EM analyses of mice of indicated genotypes . Scale bar is 10 μm for panels 1 - 4 , with fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3 . F88W PrP ( scale bar , 5 μm ) .

O O O B-TISSUE I-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O 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 B-ORGANISM O O O O B-GENEPROD O O O O O O O O

A Sections of sciatic nerve from TgPrP(S3.F88W)-14 female mice (all 610 days old) and TgPrP(S3.F88W)-35 female mice (614, 570 and 614 days old, respectively) were analysed using toluidine blue staining (upper row), with the third panels for each line showing a hypermyelinated phenotype. Scale bar in left panel represents 50 μm. The lower rows show EM analyses of mice of the indicated genotypes. Scale bar is 10 μm for panels 1-4, with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3.F88W PrP (scale bar, 5 μm).


B Western blot of sciatic nerve extracts ( 10 μg ) from mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain ( see Fig3 ) . asterisk indicates a greater than full - fragment present in TgPrP ( S3 . F88W ) mice , possibly corresponding to incomplete removal of N - terminal signal peptide .

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B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.


C of fibre in aged ( 492 - 608 days old ) , representing of nerve occupied by fibres versus total cross - sectional of nerve . S1 S3 . F88W alleles rescue Prnp0 / 0 nerves to a similar as WT allele , but variance P - value is greater for two Tg lines expressing TgPrP ( S3 . F88W ) allele . Unpaired , two - t - , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Sample sizes exact P - values compared with Prnp0 / 0 ( n = 9 ) were as follows : TgPrP ( WT ) n = 6 , P = 3 . 63E - 05 ; Prnp0 / 0 , n = 9 ; TgPrP ( S1 ) - 17 n = 5 , P = 3 . 54E - 05 ; TgPrP ( S1 ) - 19 n = 3 , P = 6 . 35E - 06 ; TgPrP ( S3 . F88W ) - 14 n = 3 , P = 3 . 70E - 03 ; TgPrP ( S3 . F88W ) - 35 n = 7 , P = 7 . 18E - 03 .

O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE 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 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 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 O O O O O O O O O O

C Quantification of fibre morphology in aged animals (492-608 days old), representing the percentage of the nerve occupied by fibres versus the total cross-sectional area of the nerve. The S1 and S3.F88W alleles rescue Prnp0/0 nerves to a similar percentage as the WT allele, but the variance and P-value is greater for the two Tg lines expressing the TgPrP(S3.F88W) allele. Unpaired, two-tailed t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Sample sizes and exact P-values compared with Prnp0/0 (n = 9) were as follows: TgPrP(WT) n = 6, P = 3.63E-05; Prnp0/0, n = 9; TgPrP(S1)-17 n = 5, P = 3.54E-05; TgPrP(S1)-19 n = 3, P = 6.35E-06; TgPrP(S3.F88W)-14 n = 3, P = 3.70E-03; and TgPrP(S3.F88W)-35 n = 7, P = 7.18E-03.


A , B Infected brain homogenates were PK - digested analysed by Western blot using ( A ) Sha31 antibody ( B ) after PK PNGaseF digestion using Sha31 .

O O O O B-TISSUE O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O

A, B Infected brain homogenates were PK-digested and analysed by Western blot using (A) Sha31 antibody and (B) after PK and PNGaseF digestion using Sha31.


C Conformational stability on RML - infected brain homogenates incubated with increasing concentrations of GdnHCl . best - fit alignments of data are presented . Genotypes are noted on panels . Source data are available online for this figure .

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C Conformational stability assay on RML-infected brain homogenates incubated with increasing concentrations of GdnHCl. The best-fit alignments of the data are presented. Genotypes are noted on the individual panels.Source data are available online for this figure.


Pathology of RML - infected miceA - H pattern of PrPSc deposition ( A - D ) GFAP immunostaining ( E - H ) is presented . I - P A higher power view of H & amp ; amp ; E stained hippocampus ( I - L ) thalamus ( M - P ) to reveal vacuolation is presented . Data information : of same genotype are shown within a column , incubation times of presented with RML prion isolate are shown ( days ) . Mice expressing PrP S1 S3 alleles ( central columns ) exhibit attenuated PrPSc deposition , gliosis spongy change . Pathological features of RML isolate are not altered upon sequential passage through TgPrP ( S3 . 88W ) - 35 mice ( see schematic at bottom of diagram indicating sequential passages finally , passage back into WT mice , compare data in first fourth columns ) .

O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY 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 I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-TISSUE 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 B-GENEPROD 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 B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O

Pathology of RML-infected miceA-H The pattern of PrPSc deposition (A-D) and GFAP immunostaining (E-H) is presented.I-P A higher power view of H&amp;E stained hippocampus (I-L) and thalamus (M-P) to reveal vacuolation is presented.Data information: Animals of the same genotype are shown within a column, and incubation times of the presented animals with the RML prion isolate are shown (days). Mice expressing PrP S1 and S3 alleles (central columns) exhibit attenuated PrPSc deposition, gliosis and spongy change. Pathological features of the RML isolate are not altered upon sequential passage through TgPrP(S3.88W)-35 mice (see schematic at bottom of the diagram indicating sequential passages and finally, passage back into WT mice, compare the data in first and fourth columns).


A Spot in scrapie using WT , TgPrP ( S1 ) - 17 TgPrP ( S3 . F88W ) - 14 brain homogenates ( 0 . 1 % or 0 . 01 % ) from mice infected with RML are shown in upper panel ( WT vs S1 P = 2 . 09E - 05 vs S3 P = 4 . 62E - 08 ) with numbers corrected for PrPC expression as per Table1 presented in lower panel ( WT vs S1 P = 1 . 04E - 13 vs S3 P = 3 . 39E - 20 ; two - t - , n = 3 ) .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O 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 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

A Spot counts in the scrapie cell assay using WT, TgPrP(S1)-17 and TgPrP(S3.F88W)-14 brain homogenates (0.1% or 0.01%) from mice infected with RML are shown in the upper panel (WT vs S1 P = 2.09E-05 and vs S3 P = 4.62E-08) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel (WT vs S1 P = 1.04E-13 and vs S3 P = 3.39E-20; two-tailed t-test, n = 3).


B A comparison of incubation time vs PrPC : PrPSc for mice expressing different of WT PrP ( WT , TgPrP ( WT ) , tga20 ) , S1 PrP ( TgPrP ( S1 ) - 17 , TgPrP ( S1 ) - 19 , TgPrP ( S1 ) - 39 ) S3 PrP ( TgPrP ( S3 . F88W ) - 14 , TgPrP ( S3 . F88W ) - 35 ) .

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B A comparison of incubation time vs PrPC:PrPSc ratio for mice expressing different levels of WT PrP (WT, TgPrP(WT), tga20), S1 PrP (TgPrP(S1)-17, TgPrP(S1)-19, TgPrP(S1)-39) and S3 PrP (TgPrP(S3.F88W)-14, TgPrP(S3.F88W)-35).


B ) Co - immunoprecipitation to elucidate interaction between Suv39h1 HP1 . Whole - extracts of HEK293T expressing 3xFlag - Suv39h1 or 3xFlag - ΔN - Suv39h1 were subjected to immunoprecipitation with anti - Flag tag antibody followed by immunoblotting with indicated antibodies . IgG represents heavy or

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

B) Co-immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were subjected to immunoprecipitation with the anti-Flag tag antibody followed by immunoblotting with the indicated antibodies. IgG represents the heavy or light chain


F ) Co - immunoprecipitation to elucidate interaction between Setdb1 HP1 . Whole - extracts of HEK293T expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 mutants were subjected to immunoprecipitation with anti - HP1β antibody immunocomplex was analyzed by immunoblotting with indicated antibodies .

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F) Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies.


G ) decay of HP1 interaction - defective Setdb1 mutant . mESC lines stably expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 - 3A were treated with CHX MG132 for indicated times . Stacked bar plots show summarized amounts of Setdb1 ( right ) . Data represented as mean ± SD ( n = 4 , biological replicates ) . * * p < 0 . 01 ( Tukey ' s honestly significant difference ) .

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G) Protein decay analysis of HP1 interaction-defective Setdb1 mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).


B ) expression of H3K9 MTs histone modification in HP1 - cTKO were monitored by immunoblotting . intensities were quantified using ImageJ software summarized on right . thin gray bold colored show data means of experimental replicates ( n ≥ 3 , biological replicates ) , respectively .

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B) Protein expression levels of H3K9 MTs and histone modification levels in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and means of the experimental replicates (n ≥ 3, biological replicates), respectively.


C ) Comparison of H3K9me3 between HP1 - deficient Suv39h / Setdb1 - deficient . H3K9me were quantified using ImageJ software are summarized at bottom ; horizontal represents mean of each . More than 75 nuclei were counted for each . * * p < 0 . 01 ( Tukey ' s honestly significant difference ) .

O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

C) Comparison of H3K9me3 levels between HP1-deficient cells and Suv39h/Setdb1-deficient cells. H3K9me levels were quantified using ImageJ software and are summarized at the bottom; the horizontal line represents the mean intensity of each cell. More than 75 nuclei were counted for each staining. **p < 0.01 (Tukey's honestly significant difference test).


D ) Electron microscopy of HP1 - cTKO in absence of 4OHT ( left ) of treated with 4OHT for 4 days ( right ) . Boxes represent enlarged .

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D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.


B ) Subcellular distribution of H3K9 MTs in HP1 - cTKO was examined by immunoblotting . α - Tubulin , Ezh2 , histone H3 were used as representative markers for each fraction . Stacked bar plots show abundance ratios of H3K9 MTs among three fractions ( right ) .

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B) Subcellular distribution of H3K9 MTs in the HP1-cTKO cells was examined by immunoblotting. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction. Stacked bar plots show the abundance ratios of H3K9 MTs among the three fractions (right).


B , Elucidation of - interaction between HP1 H3K9 MTs by co - immunoprecipitation . Whole - extracts of HEK293T co - expressing HA - HP1β mutants epitope - tagged of interest were subjected to immunoprecipitation with anti - HA tag antibody . immunocomplex was analyzed by immunoblotting with indicated antibodies . intensities were quantified using ImageJ software , normalized , shown on top .

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

B, Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top.


E ) Evaluation of HP1β mutants for maintaining H3K9me2 / 3 ensuring stability of H3K9 MTs . H3K9me2 / 3 of H3K9 MTs in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before after 4OHT treatment . intensities were quantified with ImageJ software are summarized on right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s ) .

O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD 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 O O O O O O O O O O O O O O O O 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) Evaluation of HP1β mutants for maintaining H3K9me2/3 and ensuring protein stability of H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


B ) expression of Jmjd1a / b in HP1 - cTKO were monitored by immunoblotting . intensities were quantified using ImageJ software summarized on right . thin gray bold colored show data mean of experimental replicates , respectively .

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

B) Protein expression levels of Jmjd1a/b in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and the mean of experimental replicates, respectively.


C ) Evaluation of HP1β mutants for ensuring stability of Jmjd1a / b . of Jmjd1a / b in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before after 4OHT treatment . intensities were quantified with ImageJ software are summarized on right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s ) .

O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O 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 O O O O O O O O O O O O O O O O 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) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a/b. Protein levels of Jmjd1a/b in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


C ) Impact of Suv39h / Setdb1 / G9a depletion on HP1 - mediated stability regulation of Jmjd1a / b . Jmjd1a / b were destabilized in mESCs lacking Suv39h / Setdb1 / G9a , even though HP1 was present .

O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O

C) Impact of Suv39h/Setdb1/G9a depletion on HP1-mediated protein stability regulation of Jmjd1a/b. Jmjd1a/b were destabilized in mESCs lacking Suv39h/Setdb1/G9a, even though HP1 was present.


D ) Functional evaluation of H3K9me2 / 3 for confining HP1 to . Subcellular localizations of HP1α , β γ in TT2 mESCs lacking Suv39h / Setdb1 / G9a were compared . α - Tubulin , Ezh2 , histone H3 were used as representative markers for each fraction .

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

D) Functional evaluation of H3K9me2/3 for confining HP1 to chromatin. Subcellular localizations of HP1α, β and γ in TT2 and mESCs lacking Suv39h/Setdb1/G9a were compared. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction.


Growth suppression in cancer after 72h of treatment with APR - 246 + / - MRP1 inhibitor MK - 571 as shown by WST - 1 ( n ≥ 3 , n shown in Table S1 ) . Cross in indicates no data available .

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Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available.


Growth suppression in human dermal fibroblasts ( HDF ) by WST - 1 after 72h of with APR - 246 + / - MK - 571 ( n = 3 ) .

Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3).


Mean IC50 values ( µM ) for APR - 246 + / - 20 μM MK - 571 of 21 lines . * p < 0 . 0001 , Wilcoxon . Each dot represents one . Mean IC50 values n shown in Table S1 . ZIP synergy scores of most synergistic sub - grouped according to TP53 gene status for 21 lines . Each dot represents one . Scores n are shown in Table S1 .

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

Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1.


Sub - G1 content of OVCAR - 3 ( R248Q ) after APR - 246 + / - MK - 571 treatment as assessed by propidium iodide at 48h ( n = 3 ) .

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

Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3).


OVCAR - 3 confluency by IncuCyte ® during 72h of treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3).


Growth suppression in HCT116 WT ( n = 4 ) R248W ( n = 3 ) after 72h treatment with APR - 246 + / - MRP1 inhibitor Reversan , as shown by WST - 1 .

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Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1.


Western blot of MRP1 ( Signaling . ) , p53 ( DO - 1 ) GAPDH of HCT116 WT R248W 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53 .

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Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53.


Growth suppression ( WST - 1 ) of HCT116 WT R248W transfected with MRP1 siRNA ( n ≥ 5 n ≥ 2 respectively ) after 48h APR - 246 treatment . Indicated values are average of four siRNAs targeting MRP1 two negative control siRNAs . Data n of siRNAs are shown in Fig S1J , data is also part of Fig 7B .

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

Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B.


Western blot of MRP1 ( Signaling . ) , p53 ( DO - 1 ) GAPDH of HCT116 WT R248W 72h after transfection with empty vector ( EV ) MRP1 expression vector .

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Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector.


Growth suppression ( WST - 1 ) of HCT116 WT R248W transfected with empty vector ( EV ) MRP1 vector after 72h APR - 246 treatment ( n = 4 ) . Data is also part of Fig S7E .

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

Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.


Growth of Eso26 xenografts ( R248W mutant TP53 ) in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 MK - 571 for 16 days . p values determined by one - way ANOVA with Tukey ' s correction at day 18 , n is indicated in figure . Kaplan - Meier plot of time to reach 1400 mm3 volume in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 MK - 571 for 16 days . Mice that were culled at ethical endpoints prior to reaching a volume of > 1400mm3 are marked as censored observations . p values were determined by Log - rank ( Mantell - Cox ) between different treatment groups . n is indicated in figure .

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

Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.   Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of >1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.


of Ki67 immunohistochemistry of Eso26 xenograft tumors at an early timepoint ( 5 days ) or post treatment / late timepoint ( > 22 days ) after treatment initiation with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) . Dots indicate tumors .

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Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint (5 days) or post treatment / late timepoint (>22 days) after treatment initiation with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Dots indicate individual tumors.


of caspase 3 immunohistochemistry of Eso26 xenograft tumors in same tumors as shown in Fig . 2C . Dots indicate tumors .

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Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig. 2C. Dots indicate individual tumors.


Representative of hematoxylin / eosin ( H & E ) immunostaining of Ki67 , caspase 3 p53 , post treatment / late timepoint ( > 22 days after treatment initiation ) with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) .

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

Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post treatment / late timepoint (>22 days after treatment initiation) with APR-246 (50mg/kg) +/- MK-571 (50mg/kg).


Growth suppression determined by ATP - CTG in colorectal cancer - derived organoids ( colo - PDO ) after treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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Growth suppression determined by the ATP-based CTG assay in colorectal cancer patient-derived organoids (colo-PDO) after treatment with APR-246 +/- MK-571 (n = 3).


IC50 values ( µM ) APR - 246 + / - 20 μM MK - 571 in indicated PDOs . * p = 0 . 04 , paired t ( n = 3 except colo - PDO2 eso - PDO3 where n = 2 ) .

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IC50 values (µM) APR-246 +/- 20 μM MK-571 in indicated PDOs. *p = 0.04, paired t test (n = 3 except colo-PDO2 and eso-PDO3 where n = 2).


Organoid ( in black , left y - axis ) PI ( in red , right y - axis ) of colo - PDO1 colo - PDO2 as determined by image 72h after treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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Organoid area (in black, left y-axis) and PI intensity (in red, right y-axis) of colo-PDO1 and colo-PDO2 as determined by image analysis 72h after treatment with APR-246 +/- MK-571 (n = 3).


14C accumulation ( cpm / mg x ml - 1 ) in 11 lines after 24h 14C - APR - 246 + / - MK - 571 treatment ( n ≥ 3 ) . * p = 0 . 0006 , paired t - . 14C - accumulation n of lines as shown in Table S3 .

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14C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3.


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 ( WT R248W ) after 24h + treatment with 14C - APR - 246 + / - MK - 571 or Reversan ( n ≥ 3 , each dots in figure ) .

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14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT R248W ( n ≥ 3 ≥ 2 ) transfected with MRP1 siRNAs after 24h of 14C - APR - 246 treatment . Indicated values represent four MRP1 siRNAs two negative control ( Ctrl ) siRNAs , where each dot is an siRNA . Mean of 14C - accumulation after treatment n with siRNA sequences is shown in Table S4 . * p = 0 . 04 for R248W p = 0 . 06 , Wilcoxon matched pairs signed rank .

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14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.


APR - 246 content in OVCAR - 3 ( TP53 R248Q ) 24h after treatment with APR - 246 + / - MK - 571 , ( n = 3 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± . See also Appendix Figure S4 .

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APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Glutathione - conjugated MQ ( GS - MQ ) in OVCAR - 3 after 24h treatment with APR - 246 + / - MK - 571 ( n = 2 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± . See also Appendix Figure S4 .

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Glutathione-conjugated MQ (GS-MQ) in OVCAR-3 cells after 24h treatment with APR-246 +/- MK-571 (n = 2). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Amount of GS - MQ at indicated timepoints after incubation of GS - MQ + / - NAC ( n = 2 ) . See Fig . EV4 for chemical reaction . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± . See also Appendix Figure S4 .

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

Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Amount of GS - MQ by LC - MS ( green , left axis ) NAC - MQ ( purple , right axis ) over time after incubation of GS - MQ with NAC ( n = 2 ) . Values on y - axes are not comparable because GS - MQ NAC - MQ have different response signals on MS . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± . See also Appendix Figure S4 .

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

Amount of GS-MQ by LC-MS (green line, left axis) and NAC-MQ (purple line, right axis) over time after incubation of GS-MQ with NAC (n = 2). Values on y-axes are not comparable because GS-MQ and NAC-MQ have different response signals on MS. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Mean IC50 ( µM ) values of APR - 246 in 17 different lines ( n ≥ 3 ) as shown by WST - 1 . * p = 0 . 03 , Mann Whitney . Mean IC50 ( µM ) values for APR - 246 treatment n for lines are also shown in Fig 1C listed in Table S1 . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

Mean IC50 (µM) values of APR-246 in 17 different cell lines (n ≥ 3) as shown by WST-1. *p = 0.03, Mann Whitney test. Mean IC50 (µM) values for APR-246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Box - - whisker plot of Pearson correlations between PRIMA - 1 - under - - curve ( AUC ) of 225 different metabolites from DepMap portal . Central band indicates median , boxes indicate 25th 75th percentile , whiskers show 2 . 5th 97 . 5th percentile outlier metabolites . High GSH GSSG ( in red ) correlate with low PRIMA - 1 sensitivity . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-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 B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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

Box-and-whisker plot of Pearson correlations between PRIMA-1 area-under-the-curve (AUC) and the levels of 225 different metabolites from the DepMap portal. Central band indicates median, boxes indicate 25th and 75th percentile, and whiskers show 2.5th and 97.5th percentile outlier metabolites. High GSH and GSSG (in red) correlate with low PRIMA-1 sensitivity. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG ) as shown by glutathione reductase ( GR ) re - cycling in H1299 R175H ( tet - off ) ( + / - doxycycline ) H1299 - / - 24h after seeding ( n = 3 ) . Indicated values are fold change compared to mutant TP53 . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG ) as shown by GR re - cycling in untreated HCT116 ( R248W , WT - / - ) 48h after seeding ( n = 3 , 5 or 2 respectively ) . Indicated values are fold change to mutant TP53 . Part of this data is shown in Fig . EV4 . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Thiol tracker by flow cytometry 48h after seeding of indicated untreated ( n = 2 ) Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Correlation between 14C accumulation ( cpm / mg x ml - 1 ) after treatment with 25 µM 14C - APR - 246 IC50 ( µM ) values for APR - 246 in 11 lines ( n ≥ 3 ) ( r = - 0 . 66 , p = 0 . 03 , Pearson correlation ) . Each dot represents one , 14C accumulation data are shown in Fig 3B values n for lines are listed in Table S1 S3 . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


ABCC1 mRNA Expression , RSEM ( Batch normalized from Illumina HiSeq _ RNASeqV2 ) in lung cancer ( luad & lusc studies , * p < 0 . 0001 ) colon adenocarcinoma ( coad study , * p = 0 . 005 ) grouped into having no alterations or putative driver missense mutations in TP53 . Statistical by Mann Whitney , n is indicated in figure . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

ABCC1 mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) in lung cancer (luad & lusc studies, *p<0.0001) and colon adenocarcinoma (coad study, *p=0.005) grouped into having no alterations or putative driver missense mutations in TP53. Statistical analysis by Mann Whitney test, n is indicated in the figure. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


14C accumulation ( cpm / mg x ml - 1 ) in H1299 R175H ( + / - doxycycline ) H1299 - / - after 24h treatment with 14C - APR - 246 ( n = 2 ) . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Growth suppression in H1299 R175H ( + / - doxycycline ) H1299 - / - treated for 72h with APR - 246 as shown by WST - 1 ( n = 3 ) . Data information : TP53 status is indicated for each . Data are represented as mean ± . See also Figure EV5 Appendix Figure S5 .

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

Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG per 106 ) , as determined by GR re - cycling after 24h MK - 571 treatment in six different lines ( n ≥ 3 , except HCT116 WT n = 2 ) . * p = 0 . 014 , Paired t . Each dot represents one , see Fig . EV6A for lines n .

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

Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n.


Intracellular oxidized glutathione ( GSSG per 106 ) as shown by LC - MS in OVCAR - 3 ( R248Q TP53 ) at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular reduced glutathione ( GSH per 106 ) as shown by LC - MS in OVCAR - 3 at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Western blot of MRP1 ( Signaling ) , xCT GAPDH of OVCAR - 3 treated with APR - 246 + / - MK - 571 for 24h .

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h.


Western blot of MRP1 ( Signaling ) , xCT GAPDH of HCT116 WT R248W treated with APR - 246 + / - MK - 571 for 24h .

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

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h.


Intracellular cystine ( CySS per 106 ) as shown by LC - MS in OVCAR - 3 at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular cysteine ( Cys per 106 ) as shown by LC - MS in OVCAR - 3 at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT R248W mutant TP53 + - transfected with MRP1 or xCT siRNA following 24h of 14C - APR - 246 treatment + / - MK - 571 . Indicated values are mean values from four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) two control siRNAs ( n ≥ 3 ) . Mean 14C - accumulation after treatment n of siRNAs is shown in Table S4 . Data for control MRP1 siRNA after 14C - APR - 246 treatment is also shown in Fig 3D .

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

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D.


Growth suppression in HCT116 WT R248W transfected with MRP1 or xCT siRNA after 48h of APR - 246 treatment as shown by WST - 1 . Indicated values are mean values of four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) two control siRNAs ( n ≥ 3 ) . Data for control MRP1 siRNA after APR - 246 single treatment are also included in Fig 1I . Values n for siRNA shown in Fig . S7C .

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

Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C.


Total intracellular glutathione ( GSH + GSSG per 106 ) in HCT116 WT as shown by GR re - cycling 48h post transfection of siRNA . Indicated values are means from four MRP1 siRNAs ( n = 3 ) , two xCT siRNA ( n = 2 - 3 ) two control siRNA ( n = 2 - 3 ) .

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

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3).


Total intracellular glutathione ( GSH + GSSG per 106 ) in HCT116 WT R248W measured by GR re - cycling after 24h of MK - 571 or sulfasalazine ( SSZ ) treatment ( n ≥ 3 , except SSZ where n = 2 , n indicated by dots ) .

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

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT R248W at 24h treatment with 14C - APR - 246 , combined with MK - 571 or sulfasalazine ( SSZ ) , ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots ) . Data for 14C - APR - 246 + / - MK - 571 is also shown in Fig 3C S3D .

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

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D.


of HCT116 WT R248W assessed by DCTM at 24h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) , or untreated ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots )

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

Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots)


OVCAR - 3 ( R248Q TP53 ) confluence by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalzine ( SSZ ) ( n = 3 ) . Part of data are shown in Fig 1G . Growth suppression in OVCAR - 3 as determined by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) depicted as heatmaps ( n = 3 ) .

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

OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3).


a , 2 - bp deletion identified in exon 9b of 1 . b , T - to - A nonsense mutation in exon 4 of 2 ( stop codon is underlined ) . c , Upper panel , intronic mutation in 3 ; lower panel , sequencing of RT - PCR product in 3 , showing exon - 6 skipping . d , Upper panel , G - to - A point mutation at splice - donor site in intron 5 of 6 ; lower panel , sequencing of RT - PCR product in 6 , showing a 6 - bp insertion at junction between exons 5 6 . inserted ( underlined ) is derived from first six nucleotides of intron 5 ( underlined ) , which creates a stop codon ( dotted ) . Most probably , nucleotides 7 8 , GT ( double underlined ) , in intron 5 are alternatively recognized as a splice - donor site . e , Upper panel , 10 - bp deletion in 9 . This mutation deletes four nucleotides from intron 5 six from exon 6 . Lower panel , of RT - PCR product in 9 reveals deletion of 10 nucleotides at 5 ′ end of exon 6 . ' AG ' ( double underlined ) in exon 6 immediately after genomic deletion appears to be recognized as an alternative splice - acceptor site .

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

a, The 2-bp deletion identified in exon 9b of patient 1. b, The T-to-A nonsense mutation in exon 4 of patient 2 (stop codon is underlined). c, Upper panel, intronic mutation in patient 3; lower panel, sequencing of the RT-PCR product in patient 3, showing exon-6 skipping. d, Upper panel, The G-to-A point mutation at the splice-donor site in intron 5 of patient 6; lower panel, sequencing of the RT-PCR product in patient 6, showing a 6-bp insertion at the junction between exons 5 and 6. The inserted sequence (underlined) is derived from the first six nucleotides of intron 5 (underlined), which creates a stop codon (dotted). Most probably, nucleotides 7 and 8, GT (double underlined), in intron 5 are alternatively recognized as a splice-donor site. e, Upper panel, the 10-bp deletion in patient 9. This mutation deletes four nucleotides from intron 5 and six from exon 6. Lower panel, sequence of the RT-PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5′ end of exon 6. The 'AG' (double underlined) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice-acceptor site.


Figure 2 Representation of mutant LAMP - 2b . ' Normal ' represents LAMP - 2b - acid . Because 1 , who developed full DD symptoms , has a mutation in exon 9b , LAMP - 2b isoform is most probably responsible for DD . Residue 265 , one of putative disulphide - bond - creating cysteines , two glycosylation sites are lost by exon - 6 skipping in 3 , which probably results in a significant change in secondary structure . Black boxes represent loops created between two cystein residues flanking each of four loop regions ; Y depicts a glycosylation site ; hatched boxes show altered caused by frameshift mutations ; dashes represent missing due to exon - 6 skipping in 3 . of other mutant lack transmembrane domain ( grey bar ) because of nonsense or frameshift mutations . Thus , those molecules cannot function as lysosomal ' ' .

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

Figure 2 Representation of the mutant LAMP-2b. 'Normal' represents the LAMP-2b amino-acid sequence. Because patient 1, who developed full DD symptoms, has a mutation in exon 9b, the LAMP-2b isoform is most probably responsible for DD. Residue 265, one of the putative disulphide-bond-creating cysteines, and two glycosylation sites are lost by exon-6 skipping in patient 3, which probably results in a significant change in the secondary structure. Black boxes represent the loops created between two cystein residues flanking each of the four loop regions; Y depicts a glycosylation site; hatched boxes show altered amino acids caused by frameshift mutations; dashes represent missing amino acids due to exon-6 skipping in patient 3. All of the other mutant proteins lack the transmembrane domain (grey bar) because of nonsense or frameshift mutations. Thus, those molecules cannot function as lysosomal 'membrane' proteins.


a , Skeletal muscle . Muscle extracts from 1 - 6 10 a control were blotted labelled with antibodies against LAMP - 2 ( top panel ) β - actin ( bottom panel ) . LAMP - 2 is deficient in examined except sample from 1 that showed a trace amount of , which is probably LAMP - 2a isoform . In support of this idea , LAMP - 2b is principal LAMP - 2 species in skeletal muscle6 . Numbers denote as in Table 1 ; C , control . b , Heart . was extracted from cardiac muscle of 4 a control , blotted onto a nitrocellulose , labelled with antibodies against LAMP - 2 ( too panel ) β - actin ( bottom panel ) . LAMP - 2 is deficient also in cardiac muscle . C , control ; 4 , 4 .

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

a, Skeletal muscle. Muscle extracts from patients 1-6 and 10 and a control were blotted and labelled with antibodies against LAMP-2 (top panel) and β-actin (bottom panel). LAMP-2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein, which is probably the LAMP-2a isoform. In support of this idea, LAMP-2b is the principal LAMP-2 species in skeletal muscle6. Numbers denote patients as in Table 1; C, control. b, Heart. Protein was extracted from cardiac muscle of patient 4 and a control, blotted onto a nitrocellulose membrane, and labelled with the antibodies against LAMP-2 (too panel) and β-actin (bottom panel). LAMP-2 is deficient also in cardiac muscle. C, control; 4, patient 4.


Serial transverse sections of biopsiedmuscle from six DD , samples from a XMEA a normal control , were immunostained with antibodies against LAMP - 2 limp - I . LAMP - 2 is absent in DD ( 6 shown here ) , whereas it is present in XMEA muscle . antibody against limp - I highlighted vacuoles in both DD XMEA . LAMP - 1 antibody faintly stained both DD XMEA , suggesting that expression level of LAMP - 1 might be lower in skeletal muscle than LAMP - 2 or limp - I ( data not shown ) . muscle from without a lamp - 2 mutation also showed LAMP - 2 deficiency on immunohistochemistry similar to genetically proven DD with LAMP - 2 deficiency ( data not shown ) . immunohistochemical stains of LAMP - 2 , limp - I , LAMP - 1 in normal muscle shows numerous tiny particles accentuates type 1 fibres . H & amp ; amp ; E , haematoxylin eosin , original magnification × 150 .

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 O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Serial transverse sections of the biopsiedmuscle from six DD patients, and samples from a XMEA patient and a normal control, were immunostained with antibodies against LAMP-2 and limp-I. LAMP-2 is absent in all DD muscles (patient 6 shown here), whereas it is present in the XMEA muscle. The antibody against limp-I highlighted vacuoles in both DD and XMEA muscles. LAMP-1 antibody faintly stained both DD and XMEA muscles, suggesting that the expression level of LAMP-1 might be lower in skeletal muscle than LAMP-2 or limp-I (data not shown). The muscle from the patient without a lamp-2 mutation also showed LAMP-2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP-2 deficiency (data not shown). The immunohistochemical stains of LAMP-2, limp-I, and LAMP-1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres. H&amp;E, haematoxylin and eosin, original magnification ×150.


( B ) Representative immunofluorescence of HeLa in various mitotic stages . were extracted , fixed , co - stained for Cyclin B2 ( green ) , ACA ( red ) , ( blue ) . Scale bar , 10 µm .

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

(B) Representative immunofluorescence images of HeLa cells in various mitotic stages. Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue). Scale bar, 10 µm.


( C ) Representative immunofluorescence of HeLa that were fixed co - stained for Cyclin B2 ( green ) , mAb414 ( red ) , ( blue ) . Scale bar , 10 µm .

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

(C) Representative immunofluorescence images of HeLa cells that were fixed and co-stained for Cyclin B2 (green), mAb414 (red), and DNA (blue). Scale bar, 10 µm.


Representative immunofluorescence of HeLa treated with an indicated siRNA were extracted , fixed , co - stained for Cyclin B2 ( green ) , ACA ( red ) , ( blue ) .

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

Representative immunofluorescence images of HeLa cells treated with an indicated siRNA Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


Representative immunofluorescence of HeLa treated with an indicated various drugs ( E ) . were extracted , fixed , co - stained for Cyclin B2 ( green ) , ACA ( red ) , ( blue ) .

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Representative immunofluorescence images of HeLa cells treated with an indicated various drugs (E). Cells were extracted, fixed, and co-stained for Cyclin B2 (green), ACA (red), and DNA (blue).


( F ) HeLa were synchronized by double thymidine , then released collected at selected time points . lysate samples were resolved by SDS - PAGE analyzed by Western blotting using anti - Cyclin B1 antibody , anti - Cyclin B2 antibody , or anti - β - Actin antibody .

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(F) HeLa cells were synchronized by double thymidine, then released and collected at selected time points. Cell lysate samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B1 antibody, anti-Cyclin B2 antibody, or anti-β-Actin antibody.


( B ) Immunoprecipitation performed with two different rabbit Cyclin B2 antibodies . Nocodazole - arrested mitotic were collected lysed . Immunoprecipitation was accomplished using control IgG , anti - Cyclin B2 - 1 ( Abcam , ab185622 ) , anti - Cyclin B2 - 2 ( Proteintech , 21644 - 1 - AP ) . Immunoprecipitation samples were resolved by Western blotting using a mouse anti - Cyclin B2 antibody ( Santa Cruz , sc - 28303 ) a mouse anti - Mad2 antibody ( Santa Cruz , sc - 47747 )

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

(B) Immunoprecipitation assay performed with two different rabbit Cyclin B2 antibodies. Nocodazole-arrested mitotic cells were collected and lysed. Immunoprecipitation was accomplished using control IgG, anti-Cyclin B2-1 (Abcam, ab185622), and anti-Cyclin B2-2 (Proteintech, 21644-1-AP). Immunoprecipitation samples were resolved by Western blotting using a mouse anti-Cyclin B2 antibody ( Santa Cruz, sc-28303) and a mouse anti-Mad2 antibody (Santa Cruz, sc-47747)


( C D ) Flag immunoprecipitation . 293T were co - transfected with Flag - C - Mad2 GFP - Cyclin B1 / B2 ( C ) , with Flag - Mad1 - 1 - 215 GFP - Cyclin B1 / B2 ( D ) . After 36 h , were collected lysed , immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitates were resolved by SDS - PAGE analyzed by Western blotting using anti - Flag antibody anti - GFP

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

(C and D) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-C-Mad2 and GFP-Cyclin B1/B2 (C), and with Flag-Mad1-1-215 and GFP-Cyclin B1/B2 (D). After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-GFP antibody


( E ) GST pulldown . Purified GST , GST - Cyclin B1 - 165 - 433 , GST - Cyclin B2 - 130 - 398 - bound agarose beads were used as affinity matrices to absorb purified Trx - 6 × His - tagged C - Mad2 recombinant . Absorbed were analyzed by SDS - PAGE Coomassie Brilliant Blue

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

(E) GST pulldown assay. Purified GST, GST-Cyclin B1-165-433, and GST-Cyclin B2-130-398-bound agarose beads were used as affinity matrices to absorb purified Trx-6×His-tagged C-Mad2 recombinant protein. Absorbed proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining


( F - I ) Representative immunofluorescence of HeLa stably expressing GFP - Cyclin B2 ( F ) or GFP - Cyclin B1 ( H ) . were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h . Then were fixed co - stained for Mad2 ( red ) ( blue ) . Scale bar , 10 µm . Scatter graphs illustrating kinetochore of GFP - Cyclin B2 ( G ) or GFP - Cyclin B1 ( I ) in treated as in F H , respectively . Bars represent mean kinetochore ( ± SD ) normalized to values of siControl group . Each dot represents one ( ≥ 30 from three independent experiments )

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

(F-I) Representative immunofluorescence images of HeLa cells stably expressing GFP-Cyclin B2 (F) or GFP-Cyclin B1 (H). The cells were transfected with siControl or siMad2 for 36 h followed by treatment with nocodazole plus MG132 for 2 h. Then cells were fixed and co-stained for Mad2 (red) and DNA (blue). Scale bar, 10 µm. Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2 (G) or GFP-Cyclin B1 (I) in cells treated as in F and H, respectively. Bars represent the mean kinetochore intensity (±SD) normalized to values of the siControl group. Each dot represents one cell (≥30 cells from three independent experiments)


( B ) Flag immunoprecipitation . Flag - tagged Cyclin B2 - WT , Cyclin B2 - 4A , or Cyclin B1 were expressed in 293T purified with anti - Flag M2 beads . Subsequently , beads were incubated with mitotic HeLa lysates for 2 h , , following extensive washes , immunoprecipitatates were resolved by SDS - PAGE analyzed by Western blotting using anti - Flag antibody anti - Mad2 antibody .

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

(B) Flag immunoprecipitation assay. Flag-tagged Cyclin B2-WT, Cyclin B2-4A, or Cyclin B1 were expressed in 293T cells and purified with anti-Flag M2 beads. Subsequently, the beads were incubated with mitotic HeLa lysates for 2 h, and, following extensive washes, immunoprecipitatates were resolved by SDS-PAGE and analyzed by Western blotting using anti-Flag antibody and anti-Mad2 antibody.


( C ) Flag immunoprecipitation . 293T were co - transfected with Flag - Cyclin B2 - WT or Flag - Cyclin B2 - 4A together with GFP ( negative control ) GFP - CDK1 . After 36 h , were collected lysed , immunoprecipitation was accomplished with anti - Flag M2 beads . Immunoprecipitation samples were resolved by SDS - PAGE analyzed by Western blotting using anti - GFP antibody anti - Flag antibody .

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

(C) Flag immunoprecipitation assay. 293T cells were co-transfected with Flag-Cyclin B2-WT or Flag-Cyclin B2-4A together with GFP (negative control) and GFP-CDK1. After 36 h, the cells were collected and lysed, and immunoprecipitation was accomplished with anti-Flag M2 beads. Immunoprecipitation samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-GFP antibody and anti-Flag antibody.


( D ) Representative immunofluorescence of HeLa transfected with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . After 36 h of transfection , were treated with nocodazole for 2 h . Then were fixed co - stained for ACA ( red ) ( blue ) . ( E ) Scatter graphs illustrating kinetochore of GFP - Cyclin B2 - WT / 4A treated as in D . Bars represent mean kinetochore ( ± SD ) normalized to values of GFP - Cyclin B2 - WT . Each dot represents one ( ≥ 30 from three independent experiments ) .

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

(D) Representative immunofluorescence images of HeLa cells transfected with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for ACA (red) and DNA (blue). (E) Scatter graphs illustrating kinetochore intensity of GFP-Cyclin B2-WT/4A treated as in D. Bars represent the mean kinetochore intensity (±SD) normalized to the values of GFP-Cyclin B2-WT. Each dot represents one cell (≥30 cells from three independent experiments).


( A ) HeLa were transfected with siControl or siCyclin B2 . At 36 h after transfection , were collected lysed . samples were resolved by SDS - PAGE analyzed by Western blotting using anti - Cyclin B2 antibody , anti - Cyclin B1 antibody , anti - tubulin

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

(A) HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were collected and lysed. The samples were resolved by SDS-PAGE and analyzed by Western blotting using anti-Cyclin B2 antibody, anti-Cyclin B1 antibody, and anti-tubulin antibody


( B ) Representative stills illustrating mitotic progression in mCherry - H2B expressed treated with Control siRNA or Cyclin B2 siRNA . were acquired at indicated time points after start of NEBD . arrow indicates lagging

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

(B) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells treated with Control siRNA or Cyclin B2 siRNA. Images were acquired at the indicated time points after the start of NEBD. The arrow indicates lagging chromosomes


( C ) Bar graph illustrating of with normal anaphase or with anaphase lagging chromosomes in treated as in B . Bars are means ± SD ( ≥ 30 from three independent experiments )

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

(C) Bar graph illustrating the percentage of cells with normal anaphase or with anaphase lagging chromosomes in cells treated as in B. Bars are means ±SD (≥30 cells from three independent experiments)


( D ) Scatter graph of time from NEBD to beginning of anaphase in treated as in B ( n = 30 )

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(D) Scatter graph of the time from NEBD to the beginning of anaphase in cells treated as in B (n = 30 cells)


( E ) Representative stills illustrating mitotic progression in mCherry - H2B expressed depleted of endogenous Cyclin B2 rescued with GFP - Cyclin B2 - WT or GFP - Cyclin B2 - 4A . were acquired at indicated time points after start of

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

(E) Representative stills illustrating mitotic progression in mCherry-H2B expressed cells depleted of endogenous Cyclin B2 and rescued with GFP-Cyclin B2-WT or GFP-Cyclin B2-4A. Images were acquired at the indicated time points after the start of NEBD


( F ) Bar graph illustrating of with normal anaphase with anaphase lagging chromosomes in treated as in E . Bars are means ± SD ( ≥ 30 from three independent experiments )

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

(F) Bar graph illustrating the percentage of cells with normal anaphase and cells with anaphase lagging chromosomes in cells treated as in E. Bars are means ±SD (≥30 cells from three independent experiments)


Representative immunofluorescence of HeLa transfected with siControl or siCyclin B2 . After 36 h of transfection , were treated with nocodazole for 2 h . Then were fixed co - stained for Mad2 ( green ) in D , ACA ( red ) , ( blue ) Representative immunofluorescence of HeLa co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , were treated with nocodazole for 2 h . Then were fixed co - stained for Mad2 ( red ) in ( blue )

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

Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (green) in D, ACA (red), and DNA (blue) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained for Mad2 (red) in DNA (blue)


Scatter graphs illustrating kinetochore of Mad2 ( treated as in A B Bars represent mean kinetochore ( ± SD ) normalized to values of siControl

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Scatter graphs illustrating kinetochore intensity of Mad2 ( cells treated as in A and B Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


Representative immunofluorescence of HeLa transfected with siControl or siCyclin B2 . After 36 h of transfection , were treated with nocodazole for 2 h . Then were fixed co - stained fo Cdc20 ( green ) in D , ACA ( red ) , ( blue ) E ) Representative immunofluorescence of HeLa co - transfected with siCyclin B2 plus GFP - tagged Cyclin B2 - WT or Cyclin B2 - 4A . At 36 h post - transfection , were treated with nocodazole for 2 h . Then were fixed co - stained fo Cdc20 ( red ) in E , ( blue )

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

Representative immunofluorescence images of HeLa cells transfected with siControl or siCyclin B2. After 36 h of transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (green) in D, ACA (red), and DNA (blue) E) Representative immunofluorescence images of HeLa cells co-transfected with siCyclin B2 plus GFP-tagged Cyclin B2-WT or Cyclin B2-4A. At 36 h post-transfection, cells were treated with nocodazole for 2 h. Then cells were fixed and co-stained fo Cdc20 (red) in E, and DNA (blue)


Scatter graphs illustrating kinetochore o Cdc20 ( F , treated as in D E ) Bars represent mean kinetochore ( ± SD ) normalized to values of siControl

O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O

Scatter graphs illustrating kinetochore intensity o Cdc20 (F, cells treated as in D and E) Bars represent the mean kinetochore intensity (±SD) normalized to the values of the siControl group


( G ) Bar graph illustrating mitotic index in treated with low concentrations of nocodazole for 12

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(G) Bar graph illustrating the mitotic index in cells treated with low concentrations of nocodazole for 12 h


( H ) Immunoprecipitation performed with Cdc20 antibody . HeLa were transfected with siControl or siCyclin B2 . At 36 h after transfection , were arrested in mitosis , collected , lysed . lysates were incubated with A / G beads bound with Cdc20 antibody . Following extensive washes , immunoprecipitates were resolved by SAS - PAGE analyzed by Western blotting using anti - Cdc20 , anti - BubR1 , anti - Bub3 , anti - Mad2

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

(H) Immunoprecipitation assay performed with Cdc20 antibody. HeLa cells were transfected with siControl or siCyclin B2. At 36 h after transfection, cells were arrested in mitosis, collected, and lysed. Cell lysates were incubated with Protein A/G beads bound with Cdc20 antibody. Following extensive washes, immunoprecipitates were resolved by SAS-PAGE and analyzed by Western blotting using anti-Cdc20, anti-BubR1, anti-Bub3, and anti-Mad2 antibodies


Subcellular localization of NeoR NucNeoR T recognition of NeoR ‐ NucNeoR ‐ expressing . RCC1 . 24 were transfected with NeoR ( A ) NucNeoR ( B ) expression constructs . After 2 days , subcellular localization of was analyzed by immunofluorescence using anti c ‐ myc antibody 9E10 . While NeoR was evenly distributed in nucleus cytoplasm ( a ) , NucNeoR was confined to nucleus ( b ) . Daylight of stained are shown in ( a ' ) ( b ' ) .

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

Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR‐ and NucNeoR‐expressing cells. RCC1.24 cells were transfected with the NeoR (A) and NucNeoR (B) expression constructs. After 2 days, the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c‐myc antibody 9E10. While NeoR was evenly distributed in nucleus and cytoplasm (a), NucNeoR was confined to the cell nucleus (b). Daylight photographs of the stained cells are shown in (a') and (b').


( C ) NeoR ‐ NucNeoR ‐ transfected RCC1 . 24 were treated with 100 U / mL IFN ‐ γ for 48 h to induce MHC class II expression . Subsequently , were washed co ‐ cultured with CD4 clone 20 ‐ 4 / A4 , which recognizes a peptide derived from NeoR on HLA ‐ DP3 . GM ‐ CSF secretion by T was measured 20 h later by ELISA . Both transfectants were recognized after IFN ‐ γ treatment , indicating that directing NeoR into nucleus does not abrogate antigen presentation .

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

(C) NeoR‐ and NucNeoR‐transfected RCC1.24 cells were treated with 100 U/mL IFN‐γ for 48 h to induce MHC class II expression. Subsequently, the cells were washed and co‐cultured with the CD4 clone 20‐4/A4, which recognizes a peptide derived from NeoR on HLA‐DP3. GM‐CSF secretion by the T cells was measured 20 h later by ELISA. Both transfectants were recognized after IFN‐γ treatment, indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation.