( F ) Representative immunohistochemistry ( IHC ) images detecting FNTA , p - ERK1 / 2 and p - MEK1 / 2 in PLKO tumurs as compared to shcircFNTA xenografted tumors . Scale bars : 100 μm ( upper panels ) and 25 μm ( lower panels ) . ( G ) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA , p - ERK1 / 2 and p - MEK1 / 2 .

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(F) Representative immunohistochemistry (IHC) images detecting FNTA, p-ERK1/2 and p-MEK1/2 in PLKO tumurs as compared to shcircFNTA xenografted tumors. Scale bars: 100 μm (upper panels) and 25 μm (lower panels). (G) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA, p-ERK1/2 and p-MEK1/2.


( H ) The tumor tail vein injection model was established to observe lung metastasis ( N = 6 ) . Representative images of lung metastasis foci in gross and microscopic HE examination . Scale bars : 400 μm ( upper panels ) and 100 μm ( lower panels ) . ( I ) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group . ( J ) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group .

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(H) The tumor tail vein injection model was established to observe lung metastasis (N=6). Representative images of lung metastasis foci in gross and microscopic HE examination. Scale bars: 400 μm (upper panels) and 100 μm (lower panels). (I) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group. (J) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group.


( A ) Autophagic markers from mixed midbrain gba + / + , gba + / − , and gba − / − cultures were analyzed via western blotting using antibodies as indicated .

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(A) Autophagic markers from mixed midbrain gba+/+, gba+/−, and gba−/− cultures were analyzed via western blotting using antibodies as indicated.


( B ) Autophagic flux was analyzed in gba + / + and gba − / − midbrain neurons . Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting . β - actin was used as a loading control . ( C ) Densitometry analyzes of autophagy of ( B ) expressed as a ratio of LC3II / LC3I . Error bars , ± SEM . * p < 0 . 05 .

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

(B) Autophagic flux was analyzed in gba+/+ and gba−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. *p < 0.05.


( D ) Accumulation of p62 / SQSTM1 was analyzed via immunoblotting . β - actin was used as a loading control .

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(D) Accumulation of p62/SQSTM1 was analyzed via immunoblotting. β-actin was used as a loading control.


( E ) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO . Proteins were extracted and analyzed via western blotting and stained with anti - ubiquitin antibodies .

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(E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies.


( F ) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated . β - actin was used as a loading control .

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(F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control.


( G ) Proteasomal activity ( chymotrypsin activity ) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase . RFU , relative luciferase units . Data represent the mean ± SEM ( n = 3 , each experiment contained triplicates for each genotype ) . * p < 0 . 05 .

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(G) Proteasomal activity (chymotrypsin activity) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase. RFU, relative luciferase units. Data represent the mean ± SEM (n = 3, each experiment contained triplicates for each genotype). *p < 0.05.


( A ) Levels of soluble and insoluble α - synuclein from midbrain of P1 gba + / + and gba − / − mice . TX - 100 soluble and insoluble ( SDS / Urea ) factions were isolated and analyzed via western blotting with α - synuclein antibodies . β - actin was used as a loading control .

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(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba+/+ and gba−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control.


( B ) Sagittal brain stem sections from gba + / + , gba + / − , and gba − / − mice were stained with α - synuclein antibodies . Scale bar , 1 , 000 μm .

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(B) Sagittal brain stem sections from gba+/+, gba+/−, and gba−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.


( A ) Neurons and astrocytes from gba + / + , gba + / − , and gba − / − mice were stained with TMRM . The mean florescence intensity in mitochondria was analyzed via confocal microscopy ( n = 3 , > 32 cells analyzed / experiment ) .

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

(A) Neurons and astrocytes from gba+/+, gba+/−, and gba−/− mice were stained with TMRM. The mean florescence intensity in mitochondria was analyzed via confocal microscopy (n = 3, >32 cells analyzed/experiment).


( B ) Neurons from gba + / + and gba − / − bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy . After 1 min , 1 μM oligomycin was added followed by 1 μM FCCP ( n = 5 , three cells analyzed / experiment ) .

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(B) Neurons from gba+/+ and gba−/− bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 1 min, 1 μM oligomycin was added followed by 1 μM FCCP (n = 5, three cells analyzed/experiment).


( C ) Neurons from gba + / + and gba − / − incubated with 5 mM methyl pyruvate 5 min prior to imaging . Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy . After 90 s , 1 μM oligomycin was added , followed by 1 μM FCCP ( n = 3 , three cells analyzed / experiment ) .

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

(C) Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).


( D ) As in ( C ) , except cells were bathed in 10 mM methyl succinate , ( n = 3 , four cells / experiment ) . All data in this figure represent the mean ± SEM , * p < 0 . 05 . Neurons from gba + / + and gba − / − incubated with 5 mM methyl pyruvate 5 min prior to imaging . Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy . After 90 s , 1 μM oligomycin was added , followed by 1 μM FCCP ( n = 3 , three cells analyzed / experiment ) .

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

(D) As in (C), except cells were bathed in 10 mM methyl succinate, (n = 3, four cells/experiment). All data in this figure represent the mean ± SEM, *p < 0.05. Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).


( A ) Oxygen consumption rates of gba + / + , gba + / − , and gba − / − mixed midbrain cultures . Basal oxygen consumption was measured over 3 min . The maximal ( uncoupled ) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A ( n = 3 , three runs / experiment ) .

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

(A) Oxygen consumption rates of gba+/+, gba+/−, and gba−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment).


( B ) Complex I - NADH : Ubiquinone reductase activity from gba + / + , gba + / − , and gba − / − brains expressed as a ratio to citrate synthase ( n = 3 ) .

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(B) Complex I-NADH: Ubiquinone reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).


( C ) Complex II - III : Succinate dehydeogenase , cytochome c reductase activity from gba + / + , gba + / − , and gba − / − brains expressed as a ratio to citrate synthase ( n = 3 ) .

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

(C) Complex II-III: Succinate dehydeogenase, cytochome c reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).


( D ) Complex IV activity from gba + / + , gba + / − , and gba − / − brains expressed as a ratio to citrate synthase ( n = 3 ) .

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

(D) Complex IV activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).


( E ) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy ( n = 3 , > 16 cells / experiment ) .

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

(E) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment).


( F ) gba + / + and gba − / − cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies . All data in this figure represent the mean ± SEM , * p < 0 . 05 .

O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-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

(F) gba+/+and gba−/− cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, *p < 0.05.


( A ) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst . Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes . " A " in nucleus denotes astrocytes . Scale bar , 20 μm . ( B ) Mitochondrial morphology was blind counted from cells in ( A ) with neurons scored as MAP2 positive and astrocytes as GFAP positive . A " normal " morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild - type network . ( n = 3 ; 100 cells counted / experiment ) .

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

(A) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst. Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes. "A" in nucleus denotes astrocytes. Scale bar, 20 μm.(B) Mitochondrial morphology was blind counted from cells in (A) with neurons scored as MAP2 positive and astrocytes as GFAP positive. A "normal" morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild-type network. (n = 3; 100 cells counted/experiment).


( C ) Levels of morphology proteins from gba + / + , gba + / − , and gba − / − isolated mitochondria were analyzed via immunoblotting with the indicated antibodies .

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(C) Levels of morphology proteins from gba+/+, gba+/−, and gba−/− isolated mitochondria were analyzed via immunoblotting with the indicated antibodies.


( D ) Midbrain gba + / + , gba + / − , and gba − / − astrocytes expressing GFP - DRP1K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed ; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy . Scale bar , 20 μm ( n = 3 , > 15 cells / experiment ) . All data in this figure represent the mean ± SEM .

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

(D) Midbrain gba+/+, gba+/−, and gba−/− astrocytes expressing GFP-DRP1K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy. Scale bar, 20 μm (n = 3, >15 cells/experiment). All data in this figure represent the mean ± SEM.


( A ) Midbrain astrocytes were transfected with GFP - LC3 , treated with 100 nM bafilomycin A1 , and stained with MitoTracker Red and imaged using confocal microscopy .

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

(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy.


( B ) Midbrain gba + / + and gba − / − neurons and astrocytes were transfected with YFP - Parkin . Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy . Scale bar , 20 μm . ( C ) TMRM fluorescence intensity of cells from ( B ) was analyzed via confocal microscopy . Data represent the mean ± SEM , ( n = 3 , > 4 cells analyzed per experiment ) .

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

(B) Midbrain gba+/+ and gba−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.(C) TMRM fluorescence intensity of cells from (B) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment).


( D ) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr . PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba + / + and gba − / − neurons . Mitochondrial CI subunit NDUFS4 was used as a loading control .

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

(D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba+/+ and gba−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.


( A ) Representative images of immunofluorescence analysis ( with anti - PlxnB2 antibodies ) of the phenotype of COS7 cells transfected with wild - type or mutated PlxnB2 , compared to the collapsed phenotype induced by the cognate ligand Sema4C ( 1 μg / ml ) in cells expressing the WT receptor . ( B ) The stacked bar graph shows the fraction of collapsed PlxnB2 - expressing cells , identified as having a surface minor of 2000 μm2 ( measured by ImageJ software ) , out of 50 - 100 cells counted per each condition , from different fields and n > 3 experiments ) . The statistical analysis was performed comparing WT untreated cells with all other groups , by Fisher ' s Exact Test , which indicated a * * * * * p value < 0 . 00001 for Sema4C - treated and for G842C - PlxnB2 - expressing cells ( confirmed by ANOVA analysis on individual cell values , shown in Appendix Fig . S2 ) . By Fisher ' s test , the only other mutants significantly different from WT were R820H ( p < 0 . 001 ) and R843Q ( p < 0 . 01 ) , although ANOVA analysis on individual cell values shown in Appendix Fig . S2 didn ' t confirm such significance for other PlxnB2 variants .

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

(A) Representative images of immunofluorescence analysis (with anti-PlxnB2 antibodies) of the phenotype of COS7 cells transfected with wild-type or mutated PlxnB2, compared to the collapsed phenotype induced by the cognate ligand Sema4C (1 μg/ml) in cells expressing the WT receptor. (B) The stacked bar graph shows the fraction of collapsed PlxnB2-expressing cells, identified as having a surface minor of 2000 μm2 (measured by ImageJ software), out of 50-100 cells counted per each condition, from different fields and n>3 experiments). The statistical analysis was performed comparing WT untreated cells with all other groups, by Fisher's Exact Test, which indicated a *****p value < 0.00001 for Sema4C-treated and for G842C-PlxnB2-expressing cells (confirmed by ANOVA analysis on individual cell values, shown in Appendix Fig. S2). By Fisher's test, the only other mutants significantly different from WT were R820H (p< 0.001) and R843Q (p<0.01), although ANOVA analysis on individual cell values shown in Appendix Fig. S2 didn't confirm such significance for other PlxnB2 variants.


( A ) The graphs show the analysis of cellular viability in shPlxnB2 - tranduced agnospheres , compared to their respective scramble - transfected controls , after 24 , 48 , 72 , and 96 hours of growth in culture . Values are mean ± SD of n ≥ 3 independent experiments ( 6 technical replicates for each ) . The statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons ( Graph Pad Prism 8 . 0 . 0 ) : * * p = 0 . 0053 ; * * * * p < 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 graphs show the analysis of cellular viability in shPlxnB2-tranduced agnospheres, compared to their respective scramble-transfected controls, after 24, 48, 72, and 96 hours of growth in culture. Values are mean ± SD of n≥3 independent experiments (6 technical replicates for each). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons (Graph Pad Prism 8.0.0): **p = 0.0053; ****p < 0.0001.


( C ) After transplantation in immunodeficient mice of CUP cells described above , the volume of ADX43 - and ( D ) ADX901 - shPlxnB2 tumors was assessed , in comparison with the respective controls . Plotted values indicate the mean ± SD ( n = 8 each group ) . The statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons : * * p = 0 . 0060 ; * * * * p < 0 . 0001 .

O O O O O O O B-ORGANISM O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) After transplantation in immunodeficient mice of CUP cells described above, the volume of ADX43- and (D) ADX901-shPlxnB2 tumors was assessed, in comparison with the respective controls. Plotted values indicate the mean ± SD (n = 8 each group). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons: **p = 0.0060; ****p < 0.0001.


( A ) Time course analysis of the cellular viability of AS901 , stably overexpressing either wild - type or G842C - mutated PlxnB2 , or controls transduced with an empty vector . Plotted values are the mean ± SD of n = 3 independent experiments ( six technical replicates for each ) . The statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs . controls ( CTRL ) : at 24h * * p = 0 . 0097 , 48h * * p = 0 . 0026 , 72h * * p = 0 . 0079 , 96h * p = 0 . 0226 . PlxnB2 - G842C vs . PlxnB2 - WT : at 48h $ p = 0 . 0418 , 72h $ p = 0 . 0318 . ( B ) Tumor growth curves of ADX 901 over - expressing either PlxnB2 - G842C or PlxnB2 - WT , or mock control . Plotted values are the mean ± SD ( n = 8 each groups ) . The statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs control ( CTRL ) : at 25 days * * p = 0 . 0029 , 28 days * * p = 0 . 0028 , 32 days * * p = 0 . 0033 . PlxnB2 - G842C vs PlxnB2 - WT : at 25 days $ p = 0 . 0221 , 28 days $ p = 0 . 0234 , 32 days $ $ p = 0 . 0024 .

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

(A) Time course analysis of the cellular viability of AS901, stably overexpressing either wild-type or G842C-mutated PlxnB2, or controls transduced with an empty vector. Plotted values are the mean ± SD of n =3 independent experiments (six technical replicates for each). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs. controls (CTRL): at 24h **p = 0.0097, 48h **p = 0.0026, 72h **p = 0.0079, 96h *p = 0.0226. PlxnB2-G842C vs. PlxnB2-WT: at 48h $p = 0.0418, 72h $p = 0.0318. (B) Tumor growth curves of ADX 901 over-expressing either PlxnB2-G842C or PlxnB2-WT, or mock control. Plotted values are the mean ± SD (n = 8 each groups). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs control (CTRL): at 25 days **p = 0.0029, 28 days **p = 0.0028, 32 days **p = 0.0033. PlxnB2-G842C vs PlxnB2-WT: at 25 days $p = 0.0221, 28 days $p = 0.0234, 32 days $$p = 0.0024.


( A ) Western blotting showing basal endogenous or overexpressed PlxnB2 , either WT or G842C - mutated isoforms , in MCF7 cells . In this wide view of the hybridized membrane , it is appreciable the presence of full length molecules at 250 kDa , as well as the processed 80 kDa fragments ( reported in ref . 64 ) . Vinculin staining provided protein loading controls .

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

(A) Western blotting showing basal endogenous or overexpressed PlxnB2, either WT or G842C-mutated isoforms, in MCF7 cells. In this wide view of the hybridized membrane, it is appreciable the presence of full length molecules at 250 kDa, as well as the processed 80 kDa fragments (reported in ref. 64). Vinculin staining provided protein loading controls.


( B ) The graph shows the mean ± SD of relative increase in 24 - hour spontaneous migration across Transwell inserts of MCF7 cells ( analysed above ) over - expressing either wild - type or G842C - mutated PlxnB2 , versus mock - transfected controls ( n = 4 biological replicates ) . The statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : PlxnB2 - WT vs . mock * p = 0 . 0145 ; PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0277 ; PlxnB2 - G842C vs . mock * * * p = 0 . 0002 .

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

(B) The graph shows the mean ± SD of relative increase in 24-hour spontaneous migration across Transwell inserts of MCF7 cells (analysed above) over-expressing either wild-type or G842C-mutated PlxnB2, versus mock-transfected controls (n=4 biological replicates). The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: PlxnB2-WT vs. mock *p=0.0145; PlxnB2-G842C vs. PlxnB2-WT *p=0.0277; PlxnB2-G842C vs. mock ***p=0.0002.


( C ) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above . The graph shows the mean ± SD of values obtained in N = 3 independent experiments ( with at least three technical replicates for each ) . The statistical significance was verified by one - way ANOVA with Tukey ' s correction for multiple comparisons : * p = 0 . 0350 ; * * p = 0 . 0029 ; * * * p = 0 . 0002 . Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B .

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

(C) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above. The graph shows the mean ± SD of values obtained in N=3 independent experiments (with at least three technical replicates for each). The statistical significance was verified by one-way ANOVA with Tukey's correction for multiple comparisons: *p = 0.0350; **p = 0.0029; ***p = 0.0002. Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B.


( D ) Western blotting analysis of endogenous and overexpressed PlxnB2 ( either WT or G842C - mutated isoforms ) in CUP cells AS906 ( both full length molecules at 250 kDa and processed 80 kDa fragments ) . Vinculin staining provided protein loading controls .

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

(D) Western blotting analysis of endogenous and overexpressed PlxnB2 (either WT or G842C-mutated isoforms) in CUP cells AS906 (both full length molecules at 250 kDa and processed 80 kDa fragments). Vinculin staining provided protein loading controls.


( E ) The invasive capacity of CUP cells AS906 ( shown in previous panel ) , overexpressing WT or G842 - mutated PlxnB2 ( or mock control ) , was assessed across Matrigel - coated transwell inserts . The graph shows the mean ± SD of values obtained in N = 3 independent experiments ( including technical duplicates , in two cases ) . Below the graph , are shown representative fields of the semipermeable membranes ( additional larger size images can be found in appendix Figure S4C ) ; scale bar : 100μm . The statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : * p = 0 . 0406 ; * * p = 0 . 0045 ; * * * p = 0 . 0003 .

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

(E) The invasive capacity of CUP cells AS906 (shown in previous panel), overexpressing WT or G842-mutated PlxnB2 (or mock control), was assessed across Matrigel-coated transwell inserts. The graph shows the mean ± SD of values obtained in N=3 independent experiments (including technical duplicates, in two cases). Below the graph, are shown representative fields of the semipermeable membranes (additional larger size images can be found in appendix Figure S4C); scale bar: 100μm. The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: *p=0.0406; **p=0.0045; ***p=0.0003.


( A ) Phospho - EGFR analysis by western blotting in control and PlxnB2 - silenced AS43 cells . Images show representative results of n = 3 independent experiments , while the graph at the bottom shows the mean ± SD of normalized band quantification . The statistical significance across replicates was verified by unpaired t - test : * * * * p < 0 . 0001 . ( B ) Phospho - EGFR analysis by western blotting in AS901 and AS906 cells either mock , or overexpressing WT or G842C - mutated PlxnB2 . Images show representative results of n = 3 independent experiments , while the graphs at the bottom show the mean ± SD of normalized band quantification . The statistical significance across independent replicas was verified by one - way ANOVA followed by Tukey test : AS901 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0434 , PlxnB2 - G842C vs . mock * p = 0 . 0211 ; AS906 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0436 , PlxnB2 - G842C vs . mock * p = 0 . 0481 .

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

(A) Phospho-EGFR analysis by western blotting in control and PlxnB2-silenced AS43 cells. Images show representative results of n=3 independent experiments, while the graph at the bottom shows the mean ± SD of normalized band quantification. The statistical significance across replicates was verified by unpaired t-test: ****p<0.0001. (B) Phospho-EGFR analysis by western blotting in AS901 and AS906 cells either mock, or overexpressing WT or G842C-mutated PlxnB2. Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by one-way ANOVA followed by Tukey test: AS901 PlxnB2-G842C vs. PlxnB2-WT *p=0.0434, PlxnB2-G842C vs. mock *p=0.0211; AS906 PlxnB2-G842C vs. PlxnB2-WT *p=0.0436, PlxnB2-G842C vs. mock *p=0.0481.


( C ) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres , after 6 - hour treatment with the EGFR inhibitor cetuximab ( 50μg / ml ) . Images show representative results of n = 3 independent experiments , while the graphs at the bottom show the mean ± SD of normalized band quantification . The statistical significance across independent replicas was verified by Sidak ' s multiple comparison test between cetuximab - treated and untreated conditions , per each group : AS43 * p = 0 . 0153 , * * * p = 0 . 0001 , * * * * p < 0 . 0001 ; AS901 * p = 0 . 0201 .

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

(C) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-hour treatment with the EGFR inhibitor cetuximab (50μg/ml). Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *p=0.0153, ***p=0.0001, ****p<0.0001; AS901 *p=0.0201.


( A ) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab ( 50μg / ml ) . In the plot , each box represents values from 25th to 75th percentiles , the central band indicates the mean , and the whiskers expand from maximum to minimum values of individual cetuximab - treated experimental points ( n ≥ 9 ) , normalized to the average value of untreated cells within each group . The statistical significance across three independent experiments , each comprising ≥ 3 technical replicates , was verified by unpaired t - test multiple comparisons of cetuximab - treated and untreated conditions , per each group : * * * p = 0 . 0002 ; * * * * p < 0 . 0001 .

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 B-GENEPROD 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 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 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

(A) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab (50μg/ml). In the plot, each box represents values from 25th to 75th percentiles, the central band indicates the mean, and the whiskers expand from maximum to minimum values of individual cetuximab-treated experimental points (n≥9), normalized to the average value of untreated cells within each group. The statistical significance across three independent experiments, each comprising ≥3 technical replicates, was verified by unpaired t-test multiple comparisons of cetuximab-treated and untreated conditions, per each group: ***p=0.0002; ****p<0.0001.


( B ) The viability of AS43 , in presence ( or absence ) of cetuximab ( 50μg / ml ) , was assessed over the indicated time course , compared to values at T0 . The graph shows the mean ± SD of two independent experiments ( with quadruplicate technical replicates each ) . The statistical significance was verified through multiple comparisons by two - way ANOVA with Bonferroni correction of cetuximab - treated and untreated samples , per each time point : * * * * p < 0 . 0001 .

O O O O B-EXP_ASSAY 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 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) The viability of AS43, in presence (or absence) of cetuximab (50μg/ml), was assessed over the indicated time course, compared to values at T0. The graph shows the mean ± SD of two independent experiments (with quadruplicate technical replicates each). The statistical significance was verified through multiple comparisons by two-way ANOVA with Bonferroni correction of cetuximab-treated and untreated samples, per each time point: ****p<0.0001.


( D ) The invasive capacity of AS906 cells ( overexpressing WT or G842C - mutated PlxnB2 , or mock transfected ) was assessed as in Fig . 7E , in the presence or absence of cetuximab 50μg / ml . Images show representative results ( scale bar : 100μm ) , while the graph on the right shows the mean ± SD of n = 3 independent experiments . The statistical significance across replicates was verified by one - way ANOVA with Bonferroni correction : * * p = 0 . 0010 ; * * * p = 0 . 0005 ; * * * * p < 0 . 0001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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 O O O O 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) The invasive capacity of AS906 cells (overexpressing WT or G842C-mutated PlxnB2, or mock transfected) was assessed as in Fig. 7E, in the presence or absence of cetuximab 50μg/ml. Images show representative results (scale bar: 100μm), while the graph on the right shows the mean ± SD of n=3 independent experiments. The statistical significance across replicates was verified by one-way ANOVA with Bonferroni correction: **p= 0.0010; ***p=0.0005; ****p<0.0001.


( A ) Tandem affinity purification was used to isolate KDF1 associated proteins from SILAC - labelled keratinocytes . Precipitated proteins were resolved by SDS - PAGE and subjected to identification with LC - MS / MS . Arrows denote IgG heavy and light chains . Star denotes KDF1 . Ctrl : control . Kd : kilodalton for molecular weight markers .

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

(A) Tandem affinity purification was used to isolate KDF1 associated proteins from SILAC-labelled keratinocytes. Precipitated proteins were resolved by SDS-PAGE and subjected to identification with LC-MS/MS. Arrows denote IgG heavy and light chains. Star denotes KDF1. Ctrl: control. Kd: kilodalton for molecular weight markers.


( B - C ) HEK293 cells were transfected with empty vector or plasmids encoding HA tagged KDF1 and / or GST - tagged IKKα . Immunoprecipitation ( α - HA or α - GST ) was carried out to determine their interaction . Immunoprecipitates ( IP ) and whole cell lysate ( WCL ) were immunoblotted ( IB ) with different antibodies as indicated .

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

(B-C) HEK293 cells were transfected with empty vector or plasmids encoding HA tagged KDF1 and/or GST-tagged IKKα. Immunoprecipitation (α-HA or α-GST) was carried out to determine their interaction. Immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) with different antibodies as indicated.


( D ) Immunoprecipitation was carried out with α - KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium ( Ca ) . IPs were blotted with different antibodies as indicated . Star denotes KDF1 .

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

(D) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca). IPs were blotted with different antibodies as indicated. Star denotes KDF1.


( E ) HEK293 cells were transfected to co - express HA - tagged KDF1 with GST - tagged IKKα or its kinase dead ( KD ) mutant . IP and WCL were blotted with different antibodies as indicated .

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

(E) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its kinase dead (KD) mutant. IP and WCL were blotted with different antibodies as indicated.


( F ) HEK293 cells were transfected to co - express HA - tagged KDF1 with GST - tagged IKKα or its different truncation mutants ( EV Fig . 1B ) . IP and WCL were blotted with different antibodies as indicated . Arrow denotes antibody heavy chain .

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

(F) HEK293 cells were transfected to co-express HA-tagged KDF1 with GST-tagged IKKα or its different truncation mutants (EV Fig. 1B). IP and WCL were blotted with different antibodies as indicated. Arrow denotes antibody heavy chain.


( G ) HEK293 cells were transfected to co - express GST - tagged IKKα with HA - tagged KDF1 or its different truncation mutants ( EV Fig . 1C ) . IP and WCL were blotted with different antibodies as indicated .

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

(G) HEK293 cells were transfected to co-express GST-tagged IKKα with HA-tagged KDF1 or its different truncation mutants (EV Fig. 1C). IP and WCL were blotted with different antibodies as indicated.


( A ) H / E staining of E18 . 5 skin sections from WT , IKKα KO , and KDF1 KO mice . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 50 μm . ( B ) E18 . 5 skin sections from WT , IKKα KO , and KDF1 KO mice were immunostained with different antibodies as indicated ( Krt14 : Keratin 14 , Krt10 : Keratin 10 , Lor : Loricrin , β4 : β4 - integrin , CD104 ) . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 100 μm .

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

(A) H/E staining of E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm. (B) E18.5 skin sections from WT, IKKα KO, and KDF1 KO mice were immunostained with different antibodies as indicated (Krt14: Keratin 14, Krt10: Keratin 10, Lor: Loricrin, β4: β4-integrin, CD104). Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100 μm.


( C ) Immunoblots of WCL collected at 0 , 1 and 2 days after calcium shift with different antibodies as indicated .

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

(C) Immunoblots of WCL collected at 0, 1 and 2 days after calcium shift with different antibodies as indicated.


A ) Immunoblots of WCL from IKKα KO cells with rescued expression of HA - tagged IKKα or its mutant using different antibodies as indicated .

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

A) Immunoblots of WCL from IKKα KO cells with rescued expression of HA-tagged IKKα or its mutant using different antibodies as indicated.


B ) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated . Expression f exogenous KDF1 was induced by doxycycline ( DOX ) at different concentration .

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

B) Immunoblots of WCL from KDF1 KO cells with rescued expression of KDF1 or its mutant using different antibodies as indicated. Expression f exogenous KDF1 was induced by doxycycline (DOX) at different concentration.


C - D ) Expression of loricrin is determined by immunoblots and quantified by densitometry . The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 .

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

C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05


( C - D ) Expression of loricrin is determined by immunoblots and quantified by densitometry . The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 .

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(C-D) Expression of loricrin is determined by immunoblots and quantified by densitometry. The relative expression level of loricrin upon calcium shift in different cell types was calculated and presented as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ***, p<0.001; **, p<0.01; *, p<0.05


E ) Sections of regenerated skin developed from engrafted IKKα KO , KDF1 KO , and their rescued cells were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 50 μm .

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E) Sections of regenerated skin developed from engrafted IKKα KO, KDF1 KO, and their rescued cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=50 μm.


( A ) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated . Lo : low calcium ; Hi : high calcium .

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(A) WCL from WT and KDF1 KO cells before and after calcium shift were immunoblotted with different antibodies as indicated. Lo: low calcium; Hi: high calcium.


( B - C ) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide ( CHX ) . WCL was collected at 0 , 30 , 60 , 90 , 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody ( B ) . Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified ( C ) . Statistical analysis is conducted using 2 - way ANOVA . N = 3 ( biological replicates ) . * , p < 0 . 05 . Error bar represents S . D . ( standard deviation ) .

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(B-C) WT and KDF1 KO keratinocyte were treated with 20nM cycloheximide (CHX). WCL was collected at 0, 30, 60, 90, 120 min post CHX treatment and subjected to immunoblotting with IKKα antibody (B). Band intensity is determined by densitometry and the amount of IKKα is calculated and quantified (C). Statistical analysis is conducted using 2-way ANOVA. N=3 (biological replicates). *, p<0.05. Error bar represents S.D. (standard deviation).


( D - E ) WT and KDF1 KO keratinocyte ( right panels ) or cells transfected with plasmid encoding exogenous IKKα ( left panels ) were treated with MG132 at 10µM for 6 hours , then were subjected to immunoprecipitation using anti - ubiquitin ( Ub ) antibody . IP and WCL were analyzed by immunoblots with α - IKKα antibody ( D ) . Overall intensity of all ubiquitinated IKKα bands was determined by densitometry . Ratio of ubiquitinated IKKα was quantified and presented as bar graphs ( E ) . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * , p < 0 . 05 . Kd : kilodalton for molecular weight markers .

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(D-E) WT and KDF1 KO keratinocyte (right panels) or cells transfected with plasmid encoding exogenous IKKα (left panels) were treated with MG132 at 10µM for 6 hours, then were subjected to immunoprecipitation using anti-ubiquitin (Ub) antibody. IP and WCL were analyzed by immunoblots with α-IKKα antibody (D). Overall intensity of all ubiquitinated IKKα bands was determined by densitometry. Ratio of ubiquitinated IKKα was quantified and presented as bar graphs (E). Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). *, p<0.05. Kd: kilodalton for molecular weight markers.


( F ) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant , under both low and high calcium conditions , was examined and quantified by western

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(F) IKKα protein level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant, under both low and high calcium conditions, was examined and quantified by western blotting


( G ) PiggyBac transposon was used to ectopically express HA - tagged IKKα in KDF1 deficient cells . WCL were collected and analyzed by immunoblots with different antibodies as indicated .

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(G) PiggyBac transposon was used to ectopically express HA-tagged IKKα in KDF1 deficient cells. WCL were collected and analyzed by immunoblots with different antibodies as indicated.


( H ) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification . Skin sections from grafted tissue were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bars = 50 µm .

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(H) Overexpression of IKKα in KDF1 KO keratinocyte can restore normal skin stratification. Skin sections from grafted tissue were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bars = 50 µm.


A ) HEK293 cells were transfected to co - express KDF1 with USP7 . IP and WCL were blotted with different antibodies as indicated .

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A) HEK293 cells were transfected to co-express KDF1 with USP7. IP and WCL were blotted with different antibodies as indicated.


B ) Immunoprecipitation was carried out with α - KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium ( Ca ) , same as in Fig . 1D . IPs were blotted with different antibodies as indicated . The star denotes KDF1 .

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B) Immunoprecipitation was carried out with α-KDF1 antibody in keratinocyte cultured in medium with low or high concentration of calcium (Ca), same as in Fig. 1D. IPs were blotted with different antibodies as indicated. The star denotes KDF1.


C ) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated .

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C) WCL from WT and USP7 CRISPR KO cells were subjected to immunoblotting with different antibodies as indicated.


D ) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift .

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D) IKKα protein level in WT and USP7 KO keratinocyte were examined by immunoblotting before and after calcium shift.


E ) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours , then subjected to immunoprecipitation using anti - ubiquitin antibody . Precipitated product was analyzed by immunoblotting with IKKα antibody . Band intensity was determined by densitometry and shown as bar graphs . Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * , p < 0 . 01 . Kd : kilodalton for molecular weight markers .

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E) WT and USP7 KO keratinocyte were treated with MG132 at 10μM for 6 hours, then subjected to immunoprecipitation using anti-ubiquitin antibody. Precipitated product was analyzed by immunoblotting with IKKα antibody. Band intensity was determined by densitometry and shown as bar graphs. Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). **, p<0.01. Kd: kilodalton for molecular weight markers.


F - G ) Immunoblot of Krt10 and Loricrin ( Lor ) with WT and USP7 KO cells before and after calcium shift ( F ) . Star denotes an unspecific band in α - Loricrin blots . Band intensity was determined by densitometry and shown as bar

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F-G) Immunoblot of Krt10 and Loricrin (Lor) with WT and USP7 KO cells before and after calcium shift (F). Star denotes an unspecific band in α-Loricrin blots. Band intensity was determined by densitometry and shown as bar graphs


Statistical analysis is conducted using unpaired Student ' s t - test . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * * * , p < 0 . 0001 ; * * , p < 0 . 01 .

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Statistical analysis is conducted using unpaired Student's t-test. Error bar represents S.D. (standard deviation). N=3 (biological replicates). ****, p<0.0001; **, p<0.01.


H ) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated . Dotted lines denote dermal - epidermal boundaries . Epi : epidermis , Der : dermis . Scale bar = 100μm .

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H) Sections of engrafted skin developed from USP7 KO or control WT cells were immunostained with different antibodies as indicated. Dotted lines denote dermal-epidermal boundaries. Epi: epidermis, Der: dermis. Scale bar=100μm.


TEM image and ( B ) size distribution of pcMNPs ; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles .

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TEM image and (B) size distribution of pcMNPs; The average size of nanoparticles is represented as mean ± SD of over 250 individual particles.


Zeta potentials of bare MNPs , NH2 - MNPs and pcMNPs in water . ( D ) The magnetic response property of bare MNPs , NH2 - MNPs and MBs in 30 s .

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Zeta potentials of bare MNPs, NH2-MNPs and pcMNPs in water. (D) The magnetic response property of bare MNPs, NH2-MNPs and MBs in 30 s.


RNA binding affinity of pcMNPs analysed by native PAGE ;

RNA binding affinity of pcMNPs analysed by native PAGE;


Dispersion stability of pcMNPs in a high - salt solution ( 50 mM NaCl ) .

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Dispersion stability of pcMNPs in a high-salt solution (50 mM NaCl).


RT - PCR assays towards viral RNA extracted by different pcMNPs - based methods . RT - PCR assays amplifying ( A ) the ORF1ab region and ( B ) the N gene in pseudoviral RNA extracted by a manual protocol .

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RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.


RT - PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol , and ( B ) RT - PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol .

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RT-PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol, and (B) RT-PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol.


RT - PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT - PCR protocol , and ( B ) corresponding calibration curve .

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RT-PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT-PCR protocol, and (B) corresponding calibration curve.


RT - PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT - PCR protocol , and ( D ) corresponding calibration curve . The red line is the linear regression fit ( R2 = 0 . 998 ) .

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RT-PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT-PCR protocol, and (D) corresponding calibration curve. The red line is the linear regression fit (R2 = 0.998).


( B ) Genome browser representation of the SOX2 locus . The promoter is defined as the unmethylated region spanning the SOX2 transcriptional start site in MCF7 - control .

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(B) Genome browser representation of the SOX2 locus. The promoter is defined as the unmethylated region spanning the SOX2 transcriptional start site in MCF7-control.


Genomic distribution of ( C ) identified ZF - D3A binding sites , and ( D ) differentially methyl - ated regions ( DMRs ) between MCF7 - control and ZF - D3A + dox .

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Genomic distribution of (C) identified ZF-D3A binding sites, and (D) differentially methyl- ated regions (DMRs) between MCF7-control and ZF-D3A +dox.


( E ) Shown for all DMRs identified between ZF - D3A + dox and MCF7 - control is : ∆ mCG ( difference in the methylation level [ ratio of C base calls to total base calls for all CG dinucleotides in region , mCG / CG ] between ZF - D3A + dox and MCF7 - control ) , ZF - D3A ChIP - seq signal ( normalized ChIP - seq read density , ± 1kb flanking DMR center ) , CpG dinucleotide density ( CpG / 100bp , ± 1kb flanking DMR center ) , and classification of each DMR as promoter or non - promoter located .

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(E) Shown for all DMRs identified between ZF-D3A +dox and MCF7-control is: ∆mCG (difference in the methylation level [ratio of C base calls to total base calls for all CG dinucleotides in region, mCG/CG] between ZF-D3A +dox and MCF7-control), ZF-D3A ChIP-seq signal (normalized ChIP-seq read density, ±1kb flanking DMR center), CpG dinucleotide density (CpG/100bp, ±1kb flanking DMR center), and classification of each DMR as promoter or non-promoter located.


( F ) DNA methylation level in MCF7 - control and ZF - D3A + dox cells flanking ( ± 1 kb , 100 bp bins ) the center of ZF - D3A ChIP - seq peaks identified in ZF - D3A + dox .

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(F) DNA methylation level in MCF7-control and ZF-D3A +dox cells flanking (±1 kb, 100 bp bins) the center of ZF-D3A ChIP-seq peaks identified in ZF-D3A +dox.


( G ) Genomic distribution of unmethylated regions ( UMRs ) in MCF7 - control . ( H ) DMRs and UMRs with an intersection of ≥ 1 bp .

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(G) Genomic distribution of unmethylated regions (UMRs) in MCF7-control. (H) DMRs and UMRs with an intersection of ≥1 bp.


( A ) Line histogram of DMR methylation levels in each sample . Kernel density estimate smoothed scatterplot comparison of DMR methylation levels in MCF7 - control and ( B ) ZF - D3A + dox , or ( C ) ZF - D3A dox - wd .

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(A) Line histogram of DMR methylation levels in each sample. Kernel density estimate smoothed scatterplot comparison of DMR methylation levels in MCF7-control and (B) ZF-D3A +dox, or (C) ZF-D3A dox-wd.


( D ) Line histogram of UMR methylation levels in each sample . Scatterplot compari - son of UMR methylation levels in MCF7 - control and ( E ) ZF - D3A + dox , or ( F ) ZF - D3A dox - wd .

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(D) Line histogram of UMR methylation levels in each sample. Scatterplot compari- son of UMR methylation levels in MCF7-control and (E) ZF-D3A +dox, or (F) ZF-D3A dox-wd.


( G ) Heat - maps of DMRs ( ordered by decreasing H3K4me3 read density ) showing : DNA methylation levels ( ± 2 . 5 kb ) , differences ( ΔmCG ) , classification as promoter or non - promoter located , CpG dinucleotide density ( ± 2 . 5 kb ) , and histone modification ChIP - seq read density ( ± 2 . 5 kb ) ; ( H ) As in ( G ) but for the 6 , 000 DMRs with the lowest H3K4me3 levels , ordered by decreasing H3K4me1 read density , and DMR classification as enhancer or non - enhancer in MCF7 ( ENCODE ) .

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(G) Heat- maps of DMRs (ordered by decreasing H3K4me3 read density) showing: DNA methylation levels (±2.5 kb), differences (ΔmCG), classification as promoter or non-promoter located, CpG dinucleotide density (±2.5 kb), and histone modification ChIP-seq read density (±2.5 kb); (H) As in (G) but for the 6,000 DMRs with the lowest H3K4me3 levels, ordered by decreasing H3K4me1 read density, and DMR classification as enhancer or non-enhancer in MCF7 (ENCODE).


( A ) H3K4me3 ChIP - seq read density flanking ( ± 1 kb , 50 bp bins ) the centre of DMRs with ∆ mCG > 0 . 2 ( top ) , and H3K4me3 ChIP - seq peaks with ∆ mCG < 0 . 2 ( bottom ) .

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(A) H3K4me3 ChIP-seq read density flanking (±1 kb, 50 bp bins) the centre of DMRs with ∆mCG >0.2 (top), and H3K4me3 ChIP-seq peaks with ∆mCG <0.2 (bottom).


( B ) H3K4me3 ChIP - seq normalized read counts in DMRs with ∆ mCG > 0 . 2 , from H3K4me3 ChIP - seq in ZF - D3A no - dox and ZF - D3A + dox cells . Black line represents equal normalized read counts in ZF - D3A no - dox and ZF - D3A + dox cells ; blue line represents a linear regression of the data .

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(B) H3K4me3 ChIP-seq normalized read counts in DMRs with ∆mCG >0.2, from H3K4me3 ChIP-seq in ZF-D3A no-dox and ZF-D3A +dox cells. Black line represents equal normalized read counts in ZF-D3A no-dox and ZF-D3A +dox cells; blue line represents a linear regression of the data.


( C ) Comparison of UMR ∆ mCG and difference in H3K4me3 ChIP - seq normalized read counts between ZF - D3A no - dox and ZF - D3A + dox . Blue line represents a linear regression of the data .

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(C) Comparison of UMR ∆mCG and difference in H3K4me3 ChIP-seq normalized read counts between ZF-D3A no-dox and ZF-D3A +dox. Blue line represents a linear regression of the data.


( D ) DNA methylation levels of UMRs that intersect with H3K4me3 ChIP - seq peaks in ZF - D3A no - dox and ZF - D3A + dox cells , for genomic DNA or DNA immunoprecipitated with an anti - H3K4me3 antibody .

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(D) DNA methylation levels of UMRs that intersect with H3K4me3 ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-H3K4me3 antibody.


( E ) DNA methylation levels of UMRs that intersect with phos - pho - ser5 RNA polymerase II ChIP - seq peaks in ZF - D3A no - dox and ZF - D3A + dox cells , for genomic DNA or DNA immunoprecipitated with an anti - phospho - ser5 RNA polymerase II antibody .

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(E) DNA methylation levels of UMRs that intersect with phos- pho-ser5 RNA polymerase II ChIP-seq peaks in ZF-D3A no-dox and ZF-D3A +dox cells, for genomic DNA or DNA immunoprecipitated with an anti-phospho-ser5 RNA polymerase II antibody.


( A ) Western blot measurement of ZF - D3A protein abundance upon doxycycline induction and withdrawal .

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(A) Western blot measurement of ZF-D3A protein abundance upon doxycycline induction and withdrawal.


( B ) Box and whisker plots of the DNA methylation levels of the 121 CpG sites in the DACH1 promoter region through - out doxycycline induction and withdrawal . Whiskers indicate 1 . 5 times the interquartile range or the most extreme data point , whichever is lower .

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(B) Box and whisker plots of the DNA methylation levels of the 121 CpG sites in the DACH1 promoter region through- out doxycycline induction and withdrawal. Whiskers indicate 1.5 times the interquartile range or the most extreme data point, whichever is lower.


( C ) Average DNA methylation level at the 121 CpG sites in the DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal and cell cycle inhibition by growth in doxycycline - free media containing different cell cycle inhibitors ( Lovastatin , RO - 3306 , thymidine ) .

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(C) Average DNA methylation level at the 121 CpG sites in the DACH1 promoter region throughout doxycycline induction followed by doxycycline withdrawal and cell cycle inhibition by growth in doxycycline-free media containing different cell cycle inhibitors (Lovastatin, RO-3306, thymidine).


( D ) DMR mCG and hmCG levels in MCF7 - control and ZF - D3A + dox cells , ordered by the difference in DMR hmCG level ( hmCG / CG ) between MCF7 - control and ZF - D3A + dox cells , and heatmap representation of H3K4me3 levels in ZF - D3A + dox cells flanking ( ± 2 . 5 kb , 50 bp bins ) the centre of the DMRs .

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(D) DMR mCG and hmCG levels in MCF7-control and ZF-D3A +dox cells, ordered by the difference in DMR hmCG level (hmCG/CG) between MCF7-control and ZF-D3A +dox cells, and heatmap representation of H3K4me3 levels in ZF-D3A +dox cells flanking (±2.5 kb, 50 bp bins) the centre of the DMRs.


( E ) Heatmap of hmCG levels flanking ( ± 2 . 5 kb , 50 bp bins ) the centre of the DMRs , ordered by DMR hmCG level in MCF7 - control cells .

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(E) Heatmap of hmCG levels flanking (±2.5 kb, 50 bp bins) the centre of the DMRs, ordered by DMR hmCG level in MCF7-control cells.


( A ) Scatter plot of the difference in UMR DNA methylation levels , versus the fold change in mRNA abun - dance of UMR - associated expressed genes , between MCF7 - control and ZF - D3A + dox . Point color indicates the gene differential expression significance : red indicates FDR < 0 . 01 , black indicates FDR > 0 . 01 . Trend line ( green ) was fitted using a generalized additive model , and contour lines represent the relative density of UMRs .

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(A) Scatter plot of the difference in UMR DNA methylation levels, versus the fold change in mRNA abun- dance of UMR-associated expressed genes, between MCF7-control and ZF-D3A +dox. Point color indicates the gene differential expression significance: red indicates FDR <0.01, black indicates FDR >0.01. Trend line (green) was fitted using a generalized additive model, and contour lines represent the relative density of UMRs.


( B ) Distribution of the ratios of mRNA levels between ZF - D3A + dox and MCF7 - control , for expressed genes that have an associated UMR with a ∆ mCG > 0 . 3 . Column color indicates the class of mRNA abundance ratios between ZF - D3A + dox and MCF7 - control : blue = no decrease ; green = small decrease ; red = decrease .

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(B) Distribution of the ratios of mRNA levels between ZF-D3A +dox and MCF7-control, for expressed genes that have an associated UMR with a ∆mCG >0.3. Column color indicates the class of mRNA abundance ratios between ZF-D3A +dox and MCF7-control: blue = no decrease; green = small decrease; red = decrease.


( C ) Representative genes with high induction of promoter DNA methylation in ZF - D3A + dox and no reduction in mRNA abundance .

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(C) Representative genes with high induction of promoter DNA methylation in ZF-D3A +dox and no reduction in mRNA abundance.


Fluorescence distribution of cells stained with RNA - FISH probes targeting ( A ) GAPDH or ( B ) PSME1 mRNA before and after FACS .

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Fluorescence distribution of cells stained with RNA-FISH probes targeting (A) GAPDH or (B) PSME1 mRNA before and after FACS.


mRNA quantitation after FACS sorting of ( C ) GAPDH by RT - qPCR , and ( D ) PSME1 by RNA - seq .

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mRNA quantitation after FACS sorting of (C) GAPDH by RT-qPCR, and (D) PSME1 by RNA-seq.


DNA methylation levels of single CpGs at the promoter region of ( E ) GAPDH , and ( F ) PSME1 , for the low ( x axis ) and high ( y axis ) expressing sorted cell populations .

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DNA methylation levels of single CpGs at the promoter region of (E) GAPDH, and (F) PSME1, for the low (x axis) and high (y axis) expressing sorted cell populations.


( G ) Genome browser representation of the GAPDH ( top ) and PSME1 ( bottom ) promoter regions showing DNA methylation in MCF7 - control and low and high expressing sorted cell populations .

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(G) Genome browser representation of the GAPDH (top) and PSME1 (bottom) promoter regions showing DNA methylation in MCF7-control and low and high expressing sorted cell populations.


( H ) Distribution of per - read methylation levels ( blue line ) for selected ZF - D3A + dox WGBS reads ( ≥ 10 cytosines in the CpG context ) that align within robustly methylated DMRs ( DMR mean methylation level [ mCG / CG ] of 0 . 4 - 0 . 6 in ZF - D3A + dox and < 0 . 1 in MCF7 - control ) , and average CG methylation levels from ZF - D3A + dox WGBS data of the DMR genomic intervals covered by the selected reads ( red line ) .

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(H) Distribution of per-read methylation levels (blue line) for selected ZF-D3A +dox WGBS reads (≥10 cytosines in the CpG context) that align within robustly methylated DMRs (DMR mean methylation level [mCG/CG] of 0.4-0.6 in ZF-D3A +dox and <0.1 in MCF7-control), and average CG methylation levels from ZF-D3A +dox WGBS data of the DMR genomic intervals covered by the selected reads (red line).


A . Left : Telomerase activity measured using ddTRAP assay . The horizontal pink lines represent the threshold used in these samples for background correction . Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal . Right : Quantification of TRAP activity after normalization to SW39 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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A. Left: Telomerase activity measured using ddTRAP assay. The horizontal pink lines represent the threshold used in these samples for background correction. Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal. Right: Quantification of TRAP activity after normalization to SW39/TEL+ cells (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


B . hTR and hTERT expression , normalized to ACTB mRNA and to SW39 / TEL + . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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B. hTR and hTERT expression, normalized to ACTB mRNA and to SW39/TEL+. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


C . RNA Pol II binding at hTR promoter in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines , normalized to H3 ChIP . Control loci with , respectively , high or low transcriptional activity : c - fos promoter and subtelomeric 4qHox locus ( n = 3 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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

C. RNA Pol II binding at hTR promoter in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines, normalized to H3 ChIP. Control loci with, respectively, high or low transcriptional activity: c-fos promoter and subtelomeric 4qHox locus (n=3). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


D . H3K9me3 and H3K27me3 ( left ) or H3K4me3 and H3Ac ( right ) densities at hTR promoter in SI14 / TEL + and SI24 / ALT + , normalized to H3 ( n = 3 ) . Control loci as in C . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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D. H3K9me3 and H3K27me3 (left) or H3K4me3 and H3Ac (right) densities at hTR promoter in SI14/TEL+ and SI24/ALT+, normalized to H3 (n=3). Control loci as in C. Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


E . NFYB binding at hTR promoter in SI14 / TEL + , SI24 / ALT + , SW39 / TEL + and IMRB / ALT + cells normalized to H3 . Control loci with , respectively , high and low NFYB binding : hnRNPA1 promoter and subtelomeric 4qHox ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution unless specified otherwise . PTM , post - translational modification .

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E. NFYB binding at hTR promoter in SI14/TEL+, SI24/ALT+, SW39/TEL+ and IMRB/ALT+ cells normalized to H3. Control loci with, respectively, high and low NFYB binding: hnRNPA1 promoter and subtelomeric 4qHox (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.


A . Western blot for hTR chaperone proteins in TEL + and ALT + hybrids . β - Actin is used as loading control . Quantification of western blots is shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 2 - 4 ) . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD .

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A. Western blot for hTR chaperone proteins in TEL+ and ALT+ hybrids. β-Actin is used as loading control. Quantification of western blots is shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=2-4). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD.


B . NHP2 mRNA expression levels , normalized to ACTB mRNA and to SI14 / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD .

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B. NHP2 mRNA expression levels, normalized to ACTB mRNA and to SI14/TEL+ cells (n=3). Data information: Data are presented as mean ± SD.


C . Western blot for NHP2 in SI14 / TEL + , SI24 / ALT + and 8G12 / ALT + cell lines treated with DMSO or 10 μM MG132 for 6 h . β - Actin served as loading control . Quantifications are shown on the right , normalized to β - Actin and to SI14 / TEL + cells ( n = 5 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - test . Data information : Data are presented as mean ± SD .

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C. Western blot for NHP2 in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines treated with DMSO or 10 μM MG132 for 6 h. β-Actin served as loading control. Quantifications are shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=5). Statistical analyses were performed with Student's two-tailed unpaired t-test. Data information: Data are presented as mean ± SD.


D . hTR RNA and NHP2 mRNA expression , normalized to ACTB mRNA and to SI14 / TEL + . Data information : Data are presented as mean ± SD .

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D. hTR RNA and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+. Data information: Data are presented as mean ± SD.


E . Telomerase activity measured with ddTRAP assay in NHP2 - overexpressing 8G12 / ALT + cells , normalized to NHP2 - overexpressing SI14 / TEL + . Data information : Data are presented as mean ± SD .

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E. Telomerase activity measured with ddTRAP assay in NHP2-overexpressing 8G12/ALT+cells, normalized to NHP2-overexpressing SI14/TEL+. Data information: Data are presented as mean ± SD.


F . ( Top ) Western blot for NHP2 overexpression in 8G12 / ALT + clonal cell lines . β - Actin served as loading control . SI14 / TEL + cells are shown as reference for NHP2 expression in hybrids . ( Bottom ) Western blot for NHP2 overexpression in SI14 / TEL + and 8G12 / ALT + cells compared to empty vector control . β - Actin served as loading control . Data information : Data are presented as mean ± SD .

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F. (Top) Western blot for NHP2 overexpression in 8G12/ALT+ clonal cell lines. β-Actin served as loading control. SI14/TEL+ cells are shown as reference for NHP2 expression in hybrids. (Bottom) Western blot for NHP2 overexpression in SI14/TEL+ and 8G12/ALT+ cells compared to empty vector control. β-Actin served as loading control. Data information: Data are presented as mean ± SD.


G . hTR RNA , hTERT and NHP2 mRNA expression , normalized to ACTB mRNA and to SI14 / TEL + ( n = 3 ) . Data information : Data are presented as mean ± SD .

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G. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+ (n=3). Data information: Data are presented as mean ± SD.


H . RNA Pol II binding at hTR promoter , normalized to H3 ChIP ( n = 3 ) . See Fig . 1C legend for control loci . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons . Data information : Data are presented as mean ± SD .

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H. RNA Pol II binding at hTR promoter, normalized to H3 ChIP (n=3). See Fig. 1C legend for control loci. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons. Data information: Data are presented as mean ± SD.


A . hTR RNA , hTERT and NHP2 mRNA expression , normalized to ACTB mRNA and to 143B / TEL + cells , in a series of TEL + and ALT + human cell lines as indicated ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons .

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A. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to 143B/TEL+ cells, in a series of TEL+ and ALT+ human cell lines as indicated (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.


B . Western blot for NHP2 expression . β - Actin served as loading control . Quantification is shown on the right after normalization to β - Actin and to 143B / TEL + cells ( n = 3 ) . Data information : Data are presented as mean ± SD . Statistical analyses were performed using one - way ANOVA with post - hoc Tukey ' s test for multiple comparisons .

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B. Western blot for NHP2 expression. β-Actin served as loading control. Quantification is shown on the right after normalization to β-Actin and to 143B/TEL+ cells (n=3). Data information: Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.


A . hTR RNA expression , normalized to ACTB mRNA and to U2OS cells ( n = 3 ) . Data information : Data are shown as mean ± SD , with the red line marking the mean value .

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A. hTR RNA expression, normalized to ACTB mRNA and to U2OS cells (n=3). Data information: Data are shown as mean ± SD, with the red line marking the mean value.


B . Western blot for NHP2 expression in the early passages of hTR - expressing IMRB or U2OS cells , with β - Actin as loading control .

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B. Western blot for NHP2 expression in the early passages of hTR-expressing IMRB or U2OS cells, with β-Actin as loading control.


C . Quantification of doubling time for U2OS , U2OS - hTR , IMRB and IMRB - hTR cell lines ( n = 3 ) . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution Data information : Data are shown as mean ± SD , with the red line marking the mean value .

O O O O O O O B-CELL_LINE 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 O O O O O O O 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 doubling time for U2OS, U2OS-hTR, IMRB and IMRB-hTR cell lines (n=3). Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution Data information: Data are shown as mean ± SD, with the red line marking the mean value.


D . Western blot in U2OS and U2OS - hTR cells treated with or without Hydroxyurea ( 2 mM for 24 h ) , for the indicated proteins . β - Actin and Vinculin served as loading controls .

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

D. Western blot in U2OS and U2OS-hTR cells treated with or without Hydroxyurea (2 mM for 24 h), for the indicated proteins. β-Actin and Vinculin served as loading controls.


E . Representative images for co - localization between p - RPA32S33 ( green ) and telomeres ( red FISH probe ) . White arrows indicate co - localization events . Scale bars : 5 μm . Quantification is shown on the right . At least 50 nuclei were scored in each of the three independent experiments performed . Each dot represents the number of p - RPA32S33 - telomere co - localizations in one nucleus . Statistical analyses were performed with Student ' s two - tailed unpaired t - test with Welch ' s correction for normality of distribution . Data information : Data are shown as mean ± SD , with the red line marking the mean value .

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

E. Representative images for co-localization between p-RPA32S33 (green) and telomeres (red FISH probe). White arrows indicate co-localization events. Scale bars: 5 μm. Quantification is shown on the right. At least 50 nuclei were scored in each of the three independent experiments performed. Each dot represents the number of p-RPA32S33-telomere co-localizations in one nucleus. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution. Data information: Data are shown as mean ± SD, with the red line marking the mean value.