( D ) CCM1 incompatibility is symmetric between S . cerevisiae S . bayanus . CCM1 ORF in S . bayanus was replaced by either Sc - CCM1 ( Sb + Sc - CCM1 ) or Sb - CCM1 ( Sb + Sb - CCM1 , as a control ) coding regions . Only Sb + Sc - CCM1 exhibited respiration defects defect could not be rescued even when a whole set of S . cerevisiae chromosomes were provided ( Sb + Sc - CCM1 x Sc - ρ⁰ ) .

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(D) CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb+Sc-CCM1) or Sb-CCM1 (Sb+Sb-CCM1, as a control) coding regions. Only the Sb+Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb+Sc-CCM1 x Sc-ρ⁰).


( E ) CCM1 incompatibility probably occurred during divergence between S . bayanus common ancestor of S . cerevisiae , S . paradoxus , S . mikatae S . kudriavzevii . endogenous CCM1 in S . cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts . Only carrying S . bayanus CCM1 displayed growth defects on glycerol - containing plates .

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(E) CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates.


( A ) Sb - Ccm1 is transported efficiently to S . cerevisiae mitochondria . Total extracts ( total ) , fractions ( cyto ) or mitochondrial fractions ( mito ) were hybridized with different antibodies to detect . G6PDH ( glucose - 6 - phosphate dehydrogenase ) Cox2 served as mitochondrial markers , respectively . Both Sc - Ccm1 Sb - Ccm1 were fused with c - Myc at C - terminus expressed from a single - copy plasmid . Myc - tagged Ccm1 consistently appeared as double bands , which were probably due to some unknown modifications .

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(A) Sb-Ccm1 protein is transported efficiently to S. cerevisiae mitochondria. Total cell extracts (total), cytosolic fractions (cyto) or mitochondrial fractions (mito) were hybridized with different antibodies to detect the proteins. G6PDH (glucose-6-phosphate dehydrogenase) and Cox2 served as cytosolic and mitochondrial markers, respectively. Both Sc-Ccm1 and Sb-Ccm1 were fused with c-Myc at the C-terminus and expressed from a single-copy plasmid. The Myc-tagged Ccm1 proteins consistently appeared as double bands, which were probably due to some unknown protein modifications.


( B ) level of 15S rRNA is reduced in Sc + Sb - CCM1 . isolated from Sc + Sb - CCM1 ( labeled as Sb ) or Sc + Sc - CCM1 ( labeled as Sc ) was hybridized with gene - specific probes using Northern blots .

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(B) The level of 15S rRNA is reduced in Sc+Sb-CCM1 cells. RNA isolated from Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells was hybridized with gene-specific probes using Northern blots.


( C ) Sb - Ccm1 interacts weakly with S . cerevisiae 15S rRNA . level of Ccm1 - bound 15S rRNA was measured using quantitative PCR following mitochondrial immunoprecipitation of S . cerevisiae expressing Sc - CCM1 - 13Myc or Sb - CCM1 - 13Myc . Data was normalized to control group without Myc tag to obtain relative fold enrichment . Graph was plotted using data from three independent repeats for each p value was calculated by unpaired , two - sided student t - . Error bars indicate S . D . * * * , p value < 0 . 001 .

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(C) Sb-Ccm1 interacts weakly with S. cerevisiae 15S rRNA. The level of Ccm1-bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S. cerevisiae cells expressing Sc-CCM1-13Myc or Sb-CCM1-13Myc. Data was normalized to the control group without the Myc tag to obtain the relative fold enrichment. Graph was plotted using data from three independent repeats for each strain and p value was calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001.


( D ) Overexpressing Sb - CCM1 rescues respiration defect of Sc + Sb - CCM1 . cultures were serially - diluted spotted onto YPD ( glucose ) or YPG ( glycerol ) plates . plates were then incubated at 28 ° C until colonies were easily observed .

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(D) Overexpressing Sb-CCM1 rescues the respiration defect of Sc+Sb-CCM1 cells. Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed.


( E ) steady - state level of mtDNA - encoded is reduced in Sc + Sb - CCM1 . Immunoblotting for mitochondrial in Sc + Sb - CCM1 ( labeled as Sb ) or Sc + Sc - CCM1 ( labeled as Sc ) . Cox1 , Cox2 Cox3 are mtDNA - encoded IV subunits . Tom20 is a nucleus - encoded mitochondrial .

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(E) The steady-state level of mtDNA-encoded proteins is reduced in Sc+Sb-CCM1 cells. Immunoblotting for the mitochondrial proteins in Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells. Cox1, Cox2 and Cox3 are mtDNA-encoded complex IV subunits. Tom20 is a nucleus-encoded mitochondrial protein.


( A ) Tetrad reveals that respiration defect of Sc + Sb - CCM1 can be rescued by mutations in mitochondrial or genomes . suppressor clones were crossed with Sc + Sb - CCM1 - ρ⁰ tetrads from cross were analyzed . A non - Mendelian 4 : 0 segregation pattern for improved respiration is expected if suppressor mutation is in mtDNA ( left panel ) . Otherwise , a 2 : 2 segregation pattern would be observed if rescue effect involved a single mutation ( right panel ) .

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(A) Tetrad analysis reveals that the respiration defect of Sc+Sb-CCM1 cells can be rescued by mutations in mitochondrial or nuclear genomes. The suppressor clones were crossed with the Sc+Sb-CCM1-ρ⁰ strain and the tetrads from the cross were analyzed. A non-Mendelian 4:0 segregation pattern for improved respiration is expected if the suppressor mutation is in mtDNA (left panel). Otherwise, a 2:2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation (right panel).


( A ) Reconstituted Sb - CCM1 mutants can rescue incompatibility of Sb - CCM1 . Among 94 suppressor clones , 20 of them carry mutations in Sb - CCM1 gene . These mutants were reconstructed in Sc + Sb - CCM1 to their function . Superscripts next to Sb - CCM1 indicate respective acid substitutions numbers in parentheses indicate number of independent clones in our suppressor screen . Sc , wild - type S . cerevisiae .

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(A) Reconstituted Sb-CCM1 mutants can rescue the incompatibility of Sb-CCM1. Among 94 suppressor clones, 20 of them carry mutations in the Sb-CCM1 gene. These mutants were reconstructed in Sc+Sb-CCM1 cells to test their function. Superscripts next to Sb-CCM1 indicate the respective amino acid substitutions and numbers in parentheses indicate the number of independent clones in our suppressor screen. Sc, wild-type S. cerevisiae.


( C ) 400th residue plays a determinant role in S . cerevisiae - S . bayanus incompatibility . Both Sb - CCM1E375Q Sb - CCM1D400N mutants relieved incompatibility in S . cerevisiae , but only Sb - CCM1D400N caused respiration defects in S . bayanus . Sb - CCM1E375Q mutant was selected as a control in this experiment since it was a charged - to - polar substitution similar to Sb - CCM1D400N also appeared three times in our screen . Sb , wild - type S . bayanus .

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(C) The 400th residue plays a determinant role in the S. cerevisiae-S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus.


( D ) structural shows that intramolecular suppressor mutations were located on predicted PPR motifs . predicted structure of Ccm1 is shown at left . Blue green colors indicate two α - helices in each predicted PPR motif color indicates additional α - helices in N - terminus middle region . Red color indicates positions of suppressor mutations . Predicted PPR motifs occupy around 52 % of whole ( 453 of 871 residues ) . box at right displays a magnified version of some PPR motifs where substitutions are located .

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(D) Protein structural analysis shows that all intramolecular suppressor mutations were located on the predicted PPR motifs. The predicted protein structure of Ccm1 is shown at left. Blue and green colors indicate the two α-helices in each predicted PPR motif and white color indicates additional α-helices in the N-terminus and the middle region. Red color indicates the positions of the suppressor mutations. Predicted PPR motifs occupy around 52% of the whole protein (453 of 871 residues). The box at right displays a magnified version of some PPR motifs where the substitutions are located.


( A ) 15S rRNA are increased in suppressor clones . Total was isolated from replacement strains ( Sc + Sc - CCM1 Sc + Sb - CCM1 ) , two suppressor strains ( Sc + Sb - CCM1D400N Sc + Sb - CCM1E375Q ) one mitochondrial suppressor then subjected to quantitative PCR analyses . Relative mRNA abundance was calculated by normalizing 15S rRNA level to ATP6 mRNA level . Graph was plotted using data from five independent repeats for each p values were calculated by unpaired , two - sided student t - . Error bars indicate S . D . * * * , p value < 0 . 001 . * * , p value < 0 . 01 . Asterisks in black , comparing Sc + Sc - CCM1 with others . Asterisks in gray , comparing Sc + Sb - CCM1 with others .

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(A) 15S rRNA levels are increased in the suppressor clones. Total RNA was isolated from the replacement strains (Sc+Sc-CCM1 and Sc+Sb-CCM1), two nuclear suppressor strains (Sc+Sb-CCM1D400N and Sc+Sb-CCM1E375Q) and one mitochondrial suppressor strain and then subjected to quantitative PCR analyses. Relative mRNA abundance was calculated by normalizing the 15S rRNA level to the ATP6 mRNA level. Graph was plotted using data from five independent repeats for each strain and p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001. **, p value < 0.01. Asterisks in black, comparing Sc+Sc-CCM1 with others. Asterisks in gray, comparing Sc+Sb-CCM1 with others.


( B ) steady - state level of mtDNA - encoded is increased in suppressor clones . extracts from replacement suppressor strains were hybridized with antibodies against G6PDH , Cox1 , Cox2 or Cox3 .

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(B) The steady-state level of mtDNA-encoded proteins is increased in the suppressor clones. Protein extracts from the replacement and suppressor strains were hybridized with antibodies against G6PDH, Cox1, Cox2 or Cox3.


( B ) Seven of 12 tested PPR genes exhibit incompatibility between S . cerevisiae at least one other Saccharomyces sensu stricto species . doubling time of S . cerevisiae carrying endogenous PPR genes ( Sc ) or orthologs of other yeast species ( Sp , Sm , Sk Sb ) were measured in YPD ( glucose ) or YPG ( glycerol ) media . measurements were then normalized to doubling time of Sc to obtain relative doubling time . Graphs were plotted using data from at least three independent repeats for each . Sc , S . cerevisiae . Sp , S . paradoxus . Sm , S . mikatae . Sk , S . kudriavzevii . Sb , S . bayanus . n . d . , growth not detected . p values were calculated by unpaired , two - sided student t - . Error bars indicate S . D . * , p value < 0 . 05 . * * , p value < 0 . 01 . * * * , p value < 0 . 001 .

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(B) Seven of 12 tested PPR genes exhibit incompatibility between S. cerevisiae and at least one other Saccharomyces sensu stricto species. The doubling time of S. cerevisiae cells carrying the endogenous PPR genes (Sc) or orthologs of other yeast species (Sp, Sm, Sk and Sb) were measured in YPD (glucose) or YPG (glycerol) media. All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time. Graphs were plotted using data from at least three independent repeats for each strain. Sc, S. cerevisiae. Sp, S. paradoxus. Sm, S. mikatae. Sk, S. kudriavzevii. Sb, S. bayanus. n.d., growth not detected. p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001.


( A ) expression of AR was determined using Western blots ( overexpressing AR in J82 knocking down AR in UMUC3 ) .

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(A) The expression of AR was determined using Western blots (overexpressing AR in J82 cells and knocking down AR in UMUC3 cells).


( B ) Relative expression of top 20 up - regulated circRNAs from GEO dataset GSE92675 ( BCa tissues compared to para - tissues ) in response to AR modulation in J82 UMUC3 . # indicates an with a relatively low expression ( qRT - PCR cycle number > 35 ) , thus circRNA was excluded for further .

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(B) Relative expression of the top 20 up-regulated circRNAs from the GEO dataset GSE92675 (BCa tumor tissues compared to para-tumor tissues) in response to AR modulation in J82 and UMUC3 cells. The # indicates an RNA with a relatively low expression (qRT-PCR cycle number > 35), thus the circRNA was excluded for further analysis.


( E ) Real - time quantitative PCR ( qRT - PCR ) results show knock down efficiency for these 4 circRNAs in J82 oe - AR ( J82 - AR ) .

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(E) Real-time quantitative PCR (qRT-PCR) results show the knock down efficiency for these 4 circRNAs in J82 oe-AR (J82-AR) cells.


( F ) Transwell invasion assays showed that knock down of hsa _ circ _ 0084171 ( circ _ oo84171 ) , but not other circRNAs , can partly reverse oeAR - increased invasion in J82 . Scale bar , 100 μm .

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(F) Transwell invasion assays showed that knock down of hsa_circ_0084171 (circ_oo84171), but not the other circRNAs, can partly reverse oeAR-increased invasion in J82 cells. Scale bar, 100 μm.


( G ) MTT assays showed that shcirc _ 0084171 can partly reverse oeAR - increased cisplatin chemo - resistance ( 2 μg / ml ) in J82 . To calculate mean , value of 0 µg / ml cisplatin groups were set as 1 .

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(G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1.


( H ) qRT - PCR results showed that overexpression ( oe ) efficacy for hsa _ circ _ 0084171 ( oecirc _ 0084171 ) in UMUC3 - shAR .

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(H) The qRT-PCR results showed that the overexpression (oe) efficacy for hsa_circ_0084171 (oecirc_0084171) in UMUC3-shAR cells.


( I ) Transwell invasion assays showed that oecirc _ 0084171 can partly rescue shAR induced invasion deduction in UMUC3 . Scale bar , 100 μm .

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(I) Transwell invasion assays showed that oecirc_0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells. Scale bar, 100 μm.


( J ) MTT assays showed that oecirc _ 0084171 can partly overcome shAR effect for cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 .

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(J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.


B ) relative expression of circFNTA was elevated in multiple BCa lines ( T24 , J82 , UMUC3 , 5637 ) compared to normal bladder epithelial SVHUC as shown by qRT - PCR .

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B) The relative expression of circFNTA was elevated in multiple BCa cell lines (T24, J82, UMUC3, and 5637) compared to normal bladder epithelial cell line SVHUC as shown by qRT-PCR.


C ) expression of circFNTA GAPDH mRNA in J82 UMUC3 treated with or without RNase R was detected by qRT - PCR .

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C) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT-PCR.


D ) in situ hybridization ( FISH ) demonstrated that circFNTA was predominantly localized to cytoplasm of J82 UMUC3 . DAPI was mainly localized to cytoplasm . Scale bar , 20 μm .

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D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm.


E ) Two shcircFNTA plasmids using PLKO . 1 vector were transfected into UMUC3 . knock down efficacy was measured by qRT - PCR .

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E) Two shcircFNTA plasmids using PLKO.1 vector were transfected into UMUC3 cells. The knock down efficacy was measured by qRT-PCR.


G ) qRT - PCR results show oecircFNTA efficacy in J82 compared with PWPI vector ( left panel ) oecircFNTA or oelinearFNTA in J82 ( right panel ) .

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G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel).


Transwell invasion assays showed that shcircFNTA can decrease UMUC3 invasion ( H ) more profoundly than oelinearFNTA as compared with oevector group . Scale bar , 100 μm .

Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion (H) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.


Transwell invasion assays showed that shcircFNTA can increase J82 invasion ( I ) more profoundly than oelinearFNTA as compared with oevector group . Scale bar , 100 μm .

Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion (I) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.


J ) MTT assays showed that oecircFNTA enhances J82 chemo - resistance to cisplatin ( 0 , 1 , 2 , 5 , 10 μg / ml were tested ) . This effect was more obvious in oecircFNTA group as in oelinearFNTA group ( upper panel ) . MTT assays showed that shcircFNTA increases UMUC3 chemo - sensitivity to cisplatin ( lower panel ) .

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J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel).


K ) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with adjacent para - tissues ( N = 41 ) representative are shown . Scale bar , 250 μm .

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K) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para-tumor tissues (N=41 patients) and representative images are shown. Scale bar, 250 μm.


L ) qRT - PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para - tissues ( N = 23 ) .

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L) The qRT-PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para-tumor tissues (N=23 patients).


( A ) expression of AR FNTA ( circFNTA host gene ) were determined using Western blot showed that shAR can decrease FNTA expression in UMUC3 oeAR can increase FNTA expression in J82 .

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(A) The protein expression of AR and FNTA (circFNTA host gene) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells.


( B ) qRT - PCR results showed that mRNA of FNTA are not changed dramatically in UMUC3 with shAR J82 with oeAR .

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(B) qRT-PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR.


( C ) mRNA of 4 editing genes ( ADAR , ADAR2 , DHX9 , QKI ) were tested with manipulated AR ( UMUC3 in left panel J82 in right panel ) . Results showed ADAR2 mRNA is negatively correlated with AR expression .

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(C) The mRNA levels of 4 RNA editing genes (ADAR, ADAR2, DHX9, and QKI) were tested with manipulated AR (UMUC3 cells in left panel and J82 cells in right panel). Results showed ADAR2 mRNA is negatively correlated with AR expression.


( D ) Western blot also confirmed that ADAR2 can be negatively regulated by AR at level .

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(D) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level.


( E ) Knock down efficiency of AR ADAR2 ( shADAR2 # 1 shADAR # 2 ) in UMUC3 , as determined by qRT - PCR .

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(E) Knock down efficiency of AR and ADAR2 (shADAR2#1 and shADAR#2) in UMUC3 cells, as determined by qRT-PCR.


( F ) shADAR2 # 1 shADAR # 2 could both reverse shAR - dependent circFNTA reduction in UMUC3 .

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(F) shADAR2#1 and shADAR#2 could both reverse shAR-dependent circFNTA reduction in UMUC3 cells.


( G ) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR - dependent UMUC3 invasion reduction . Scale bar , 100 μm .

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(G) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR-dependent UMUC3 cells invasion reduction. Scale bar, 100 μm.


( H ) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR - dependent effects on cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 .

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(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.


( J ) ChIP assays confirmed that AR can directly bind to ADAR2 AREI / II ( - 572nt to - 555nt ) .

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(J) ChIP assays confirmed that AR can directly bind to ADAR2 AREI/II (-572nt to -555nt).


( K ) ChIP pull - down products were amplified by qRT - PCR reaction . IgG antibody was used as a negative control .

(K) ChIP pull-down DNA products were amplified by qRT-PCR reaction. The IgG antibody was used as a negative control.


( M ) Co - transfection of ARE Wild type ( WT ) or Mutant ( Mut ) ADAR2 promoter pGL3 - Luciferase constructs into UMUC3 with / without shAR ( left panel ) , J82 with / without oeAR ( right panel ) . luciferase was used to detect promoter activity .

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(M) Co-transfection of ARE Wild type (WT) or Mutant (Mut) ADAR2 promoter pGL3-Luciferase constructs into UMUC3 cells with/without shAR (left panel), and J82 cells with/without oeAR (right panel). The luciferase assay was used to detect promoter activity.


( A ) Western blot showed that shcircFNTA can decrease FNTA expression in UMUC3 ( upper panel ) oecircFNTA can increase FNTA expression , that this was more profound than using oelinearFNTA in J82 ( lower panel ) .

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(A) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells (upper panel) and oecircFNTA can increase FNTA expression, and that this was more profound than using oelinearFNTA in J82 cells (lower panel).


( B ) A pull - down was used to binding of circFNTA to 11 miRNAs ( predicted by online database ) in UMUC3 . results showed that miR - 328 - 5p , miR - 370 - 3p miR - 920 can be pulled - down by circFNTA .

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(B) A RNA pull-down assay was used to test the binding of circFNTA to 11 miRNAs (predicted by online database) in UMUC3 cells. The results showed that miR-328-5p, miR-370-3p and miR-920 can be pulled-down by circFNTA.


( C ) Relative miRNA expressions were quantified by qRT - PCR to miRNA overexpression efficacy .

(C) Relative miRNA expressions were quantified by qRT-PCR to test miRNA overexpression efficacy.


Transwell invasion ( D ) confirmed that only oemiR - 370 - 3p can reverse oecircFNTA induced invasion in J82 ,

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Transwell invasion assay (D) confirmed that only oemiR-370-3p can reverse oecircFNTA induced invasion in J82 cells,


MTT assays ( E ) confirmed that oemiR - 370 - 3p can reverse oecircFNTA induced cisplatin chemo - resistance ( 2 μg / ml ) in J82 . Scale bar , 100 μm .

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MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm.


Transducing miR - 370 - 3p inhibitor into UMUC3 can reverse FNTA reduction by shcircFNTA shown by Western blot ( F )

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Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot (F)


Transducing miR - 370 - 3p inhibitor into UMUC3 can reverse FNTA reduction by shcircFNTA shown by Transwell ( G ) .

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Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay (G).


( H ) MTT assays also confirmed that transducing with a miR - 370 - 3p inhibitor in UMUC3 can partly reverse cisplatin chemo - resistance ( 2 μg / ml ) reduction by shcircFNTA .

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(H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA.


( J ) transfection efficacy of oecircFNTA WT oecircFNTA Mut was tested by qRT - PCR .

(J) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT-PCR.


( K ) Transfection with oecircFNTA WT could increase J82 invasion , while oecircFNTA Mut cannot . Scale bar , 100 μm .

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(K) Transfection with oecircFNTA WT could increase J82 cells invasion, while oecircFNTA Mut cannot. Scale bar, 100 μm.


( L ) Tramsfection with oecircFNTA WT could increase J82 cisplatin chemo - resistance ( 2 μg / ml ) , while oecircFNTA Mut did not have this effect .

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(L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect.


( N ) Luciferase reporter activity after transfection of WT Mut FNTA 3 ' UTR reporter constructs into UMUC3 ( left panel ) , comparing transduced miR - 370 - 3p inhibitor vs Ctrl J82 ( right panel ) , comparing oemiR - 370 - 3p vs control PLV .

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(N) Luciferase reporter activity after transfection of WT and Mut FNTA 3'UTR reporter constructs into UMUC3 cells (left panel), comparing transduced miR-370-3p inhibitor vs Ctrl and J82 cells (right panel), and comparing oemiR-370-3p vs control PLV.


( A ) Western blots show knock - down efficiency of shFNTA in J82 UMUC3 .

(A) Western blots show the knock-down efficiency of shFNTA in J82 and UMUC3 cells.


( B ) Transwell assays indicated that shFNTA can partly reverse oecircFNTA - increased invasion in J82 UMUC3 . Quantifications are shown on right . Scale bar , 100 μm .

(B) Transwell assays indicated that shFNTA can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Quantifications are shown on the right. Scale bar, 100 μm.


( C ) MTT assays also confirmed that shFNTA can reverse oecircFNTA - increased chemo - resistance to cisplatin ( 2 μg / ml ) in J82 UMUC3 .

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(C) MTT assays also confirmed that shFNTA can reverse oecircFNTA-increased chemo-resistance to cisplatin (2 μg/ml) in J82 and UMUC3 cells.


Transwell assays ( D , with quantitations at right ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 UMUC3 . Scale bar , 100 μm .

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Transwell assays (D, with quantitations at the right) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.


MTT assays ( E ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 UMUC3 . Scale bar , 100 μm .

MTT assays (E) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.


( F ) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 did not change KRAS level significantly .

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(F) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly.


( G ) Raf - 1 RBD GST - pull - down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 ( upper panel ) , oecircFNTA can increase KRAS activity in J82 ( lower panel ) .

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(G) Raf-1 RBD GST-pull-down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells (upper panel), and oecircFNTA can increase KRAS activity in J82 cells (lower panel).


( H ) Western blots showed that shcircFNTA can decrease ERK1 / 2 MEK1 / 2 phosphorylation in UMUC3 ( left panel ) , oecircFNTA can increase their phosphorylation in J82 ( right panel ) .

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(H) Western blots showed that shcircFNTA can decrease ERK1/2 and MEK1/2 phosphorylation in UMUC3 cells (left panel), and oecircFNTA can increase their phosphorylation in J82 cells (right panel).


( A ) UMUC3 - PLKO UMUC3 - shcircFNTA were used to establish a subcutaneous nude mouse xenograft model , cisplatin ( 1 mg / kg ) was intraperitoneally administered . of tumors are shown after mice were sacrificed ( N = 6 ) . ( B ) Compared with vector ( PLKO ) group , growth rate was significantly inhibited in shcircFNTA nude mice . ( C ) weights also decreased in shcircFNTA group .

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(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6). (B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice. (C) Tumor weights also decreased in the shcircFNTA group.


( D - E ) qRT - PCR results using mouse tissues revealed that circFNTA expression was significantly reduced ( D ) , expression of its host gene FNTA was correspondently decreased in shcircFNTA group as compared to PLKO group ( E ) .

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(D-E) The qRT-PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced (D), and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group (E).


( F ) Representative immunohistochemistry ( ) detecting FNTA , p - ERK1 / 2 p - MEK1 / 2 in PLKO tumurs as compared to shcircFNTA xenografted tumors . Scale bars : 100 μm ( upper panels ) 25 μm ( lower panels ) . ( G ) of relative revealed that targeting circFNTA led to decreased expression of FNTA , p - ERK1 / 2 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 ) tail vein injection model was established to observe lung metastasis ( N = 6 ) . Representative of lung metastasis foci in gross microscopic HE examination . Scale bars : 400 μm ( upper panels ) 100 μm ( lower panels ) . ( I ) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to PLKO group . ( J ) Targeting circFNTA significantly reduced lung metastatic foci as compared to 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 + / − , 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 + / + gba − / − midbrain neurons . Neurons were treated with 100 nM bafilomycin A1 1 μM rapamycin flux assayed by comparing LC3I LC3II via western blotting . β - actin was used as a loading control . ( C ) Densitometry analyzes of autophagy of ( B ) expressed as a of LC3II / LC3I . Error bars , ± . * p < 0 . 05 .

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(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 . were extracted analyzed via western blotting 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 Proteasome Glo where activity is proportional to released luciferase . RFU , relative luciferase units . Data represent mean ± ( 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 ) of soluble insoluble α - synuclein from midbrain of P1 gba + / + gba − / − mice . TX - 100 soluble insoluble ( SDS / Urea ) factions were isolated 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 sections from gba + / + , gba + / − , 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 astrocytes from gba + / + , gba + / − , gba − / − mice were stained with TMRM . mean florescence in mitochondria was analyzed via confocal microscopy ( n = 3 , > 32 analyzed / experiment ) .

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

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

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

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

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

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(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 MitoQ10 for 48 hr stained with TMRM analyzed via confocal microscopy ( n = 3 , > 16 / experiment ) .

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

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

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(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 ) of from gba + / + , gba + / − , gba − / − isolated mitochondria were analyzed via immunoblotting with 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 + / − , gba − / − astrocytes expressing GFP - DRP1K38A were immunostained for cytochrome c mitochondrial morphologies analyzed ; concurrently were stained with TMRM analyzed via confocal microscopy . Scale bar , 20 μm ( n = 3 , > 15 / experiment ) . data in this figure represent mean ± .

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(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 , stained with MitoTracker Red imaged using confocal microscopy .

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

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(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 addition of 10 μM FCCP for 1 6 hr . PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba + / + gba − / − neurons . Mitochondrial CI subunit NDUFS4 was used as a loading control .

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

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(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 ) graphs show of viability in shPlxnB2 - tranduced agnospheres , compared to their respective scramble - transfected controls , after 24 , 48 , 72 , 96 hours of growth in culture . Values are mean ± SD of n ≥ 3 independent experiments ( 6 technical replicates for each ) . 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 .

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

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(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 of viability of AS901 , stably overexpressing either wild - type or G842C - mutated PlxnB2 , or controls transduced with an empty vector . Plotted values are mean ± SD of n = 3 independent experiments ( six technical replicates for each ) . 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 ) growth curves of ADX 901 over - expressing either PlxnB2 - G842C or PlxnB2 - WT , or mock control . Plotted values are mean ± SD ( n = 8 each groups ) . 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 .

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(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 . In this wide view of hybridized , it is appreciable presence of full molecules at 250 kDa , as well as processed 80 kDa fragments ( reported in ref . 64 ) . Vinculin provided loading controls .

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(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 ) graph shows mean ± SD of relative increase in 24 - hour spontaneous migration across Transwell inserts of MCF7 ( analysed above ) over - expressing either wild - type or G842C - mutated PlxnB2 , versus mock - transfected controls ( n = 4 biological replicates ) . 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 .

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(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 engineered as described above . graph shows mean ± SD of values obtained in N = 3 independent experiments ( with at least three technical replicates for each ) . 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 of wounded monolayers are further shown in larger in Appendix Figure S4B .

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(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 of endogenous overexpressed PlxnB2 ( either WT or G842C - mutated isoforms ) in CUP AS906 ( both full molecules at 250 kDa processed 80 kDa fragments ) . Vinculin provided loading controls .

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

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

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

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

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

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

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(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 from SILAC - labelled keratinocytes . Precipitated were resolved by SDS - PAGE subjected to identification with LC - MS / MS . Arrows denote IgG heavy chains . Star denotes KDF1 . Ctrl : control . Kd : kilodalton for molecular markers .

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

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(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 of calcium ( Ca ) . IPs were blotted with different antibodies as indicated . Star denotes KDF1 .

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

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

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

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(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 of E18 . 5 skin sections from WT , IKKα KO , 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 , 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 .

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(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 2 days after calcium shift with different antibodies as indicated .

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(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 with rescued expression of HA - tagged IKKα or its mutant using different antibodies as indicated .

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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 with rescued expression of KDF1 or its mutant using different antibodies as indicated . Expression f exogenous KDF1 was induced by doxycycline ( DOX ) at different .

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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 quantified by densitometry . relative expression level of loricrin upon calcium shift in different types was calculated presented as bar graphs . Statistical is conducted using unpaired Student ' s t - . 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


( C - D ) Expression of loricrin is determined by immunoblots quantified by densitometry . relative expression level of loricrin upon calcium shift in different types was calculated presented as bar graphs . Statistical is conducted using unpaired Student ' s t - . 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 , their rescued 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 KDF1 KO before 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 KDF1 KO keratinocyte were treated with 20nM cycloheximide ( CHX ) . WCL was collected at 0 , 30 , 60 , 90 , 120 min post CHX treatment subjected to immunoblotting with IKKα antibody ( B ) . Band is determined by densitometry amount of IKKα is calculated quantified ( C ) . Statistical 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 KDF1 KO keratinocyte ( right panels ) or 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 WCL were analyzed by immunoblots with α - IKKα antibody ( D ) . Overall of ubiquitinated IKKα bands was determined by densitometry . of ubiquitinated IKKα was quantified presented as bar graphs ( E ) . Statistical is conducted using unpaired Student ' s t - . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * , p < 0 . 05 . Kd : kilodalton for molecular 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 level in KDF1 KO keratinocyte expressing WT KDF1 or KDF1 mutant , under both low high calcium conditions , was examined 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 . WCL were collected 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 were transfected to co - express KDF1 with USP7 . IP 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 of calcium ( Ca ) , same as in Fig . 1D . IPs were blotted with different antibodies as indicated . 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 USP7 CRISPR KO 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 level in WT USP7 KO keratinocyte were examined by immunoblotting before 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 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 was determined by densitometry shown as bar graphs . Statistical is conducted using unpaired Student ' s t - . Error bar represents S . D . ( standard deviation ) . N = 3 ( biological replicates ) . * * , p < 0 . 01 . Kd : kilodalton for molecular 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 Loricrin ( Lor ) with WT USP7 KO before after calcium shift ( F ) . Star denotes an unspecific band in α - Loricrin blots . Band was determined by densitometry 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 is conducted using unpaired Student ' s t - . 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 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.


image ( B ) distribution of pcMNPs ; average of nanoparticles is represented as mean ± SD of over 250 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 pcMNPs in water . ( D ) magnetic response property of bare MNPs , NH2 - MNPs 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.


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 ) .

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


RT - PCR assays towards viral extracted by different pcMNPs - methods . RT - PCR assays amplifying ( A ) ORF1ab region ( B ) N gene in pseudoviral 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 ORF1ab region in pseudoviral extracted by automated protocol , ( B ) RT - PCR assays amplifying N gene in pseudoviral 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 N gene in pseudoviral following Conventional RT - PCR protocol , ( 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 N gene in pseudoviral following Direct RT - PCR protocol , ( D ) corresponding calibration curve . red is 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 SOX2 locus . promoter is defined as unmethylated region spanning 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 , ( D ) differentially methyl - ated regions ( DMRs ) between MCF7 - control 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 DMRs identified between ZF - D3A + dox MCF7 - control is : ∆ mCG ( difference in methylation level [ of C base calls to total base calls for CG dinucleotides in region , mCG / CG ] between ZF - D3A + dox 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 ) , 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 ) methylation level in MCF7 - control ZF - D3A + dox flanking ( ± 1 kb , 100 bp bins ) 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 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 ) histogram of DMR methylation in each sample . Kernel density estimate smoothed scatterplot comparison of DMR methylation in MCF7 - control ( 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 ) histogram of UMR methylation in each sample . Scatterplot compari - son of UMR methylation in MCF7 - control ( 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.