Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) Representative SWB assay between the C-NAT and recombinant APE1 WT and APE1 NΔ33 proteins. BSA was used as a negative control. On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa. B) Relative histogram summarizing the different binding abilities of three independent replicates towards C-NAT between BSA, APE1 WT , APE1 NΔ33 , normalized on the respective total protein staining signal (Figure S2A). C) Graph representing the summary of C-NAT-APE1 WT and C-NAT-APE1 NΔ33 kinetic parameters (kon and koff), expressed in nM, in MES buffer pH 5.5. KD values, expressed in nM, are reported (n=3). D) Representation of 2D { 15 N- 1 H} TROESY-HSQC NMR spectra recorded at 25 °C. Measurements were performed with APE1 WT alone (red), 0.6 (green) and 1:1 (blue) molar ratios of C-NAT to APE1 WT . E) Depiction of NMR-derived binding analysis on APE1 structure (PDB code: 1bix). Cyan represents deposited canonical duplex DNA (for reference only). Purple represent residues which chemical shift variations (Δ8) that, before and after inclusion of i-motif DNA, are equal or superior to 2 standard deviations from the mean. Orange represent residues that lost over 2/3 or the respective peak volume. F) Schematic representation of the three consecutive telomeric iM (3x-C-NAT) embedded in a DNA sequence used for the single molecule analysis. G) Representative kymograph of APE1 WT -mEGFP from A549 clone 31 NCE binding to the iM (3x-C-NAT) substrate. Time (expressed in s) and position (expressed in µm) are reported on the x- and y-axis, respectively. The iM position is indicated with the red arrow. H) Cumulative residence time distribution (CRTD) plot of specific binding events of APE1 WT -mEGFP to 3x-C-NAT. I) Cumulative gap time distribution (CGTD) plot of APE1 WT -mEGFP association with 3x-C-NAT under the tested conditions.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Recombinant, Negative Control, Marker, Binding Assay, Staining, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) Scheme of the damaged telomeric iM ODNs, holding an abasic site at different positions (AP14, AP16, AP17, AP20), indicated by a star. B) CD spectrum of damaged ODNs. On the x-axis, the wavelength is reported (nm), while on the y-axis the CD value (mdeg). C) CD melting profile of damaged ODNs. Temperature (expressed in °C) and CD (expressed in mdeg) are reported on the x-and y-axis, respectively. D) Graph representing the summary of iMab kinetic parameters, including Kon and Koff, towards each of the ODNs examined in MES buffer pH 5.5. KD values, expressed in nM, are reported (n=3). E) Histogram showing fluorescence change of the different ODNs dye environment (expressed in %, y-axis) caused by increasing pH (x-axis), calculated in the plateau phase of five different replicates. F) Representative SWB assays between each ODNs, as indicated upon each panel, and APE1 WT or APE1 NΔ33 proteins. BSA was used as a negative control. On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa. G) Relative histogram summarizing the different binding abilities of three independent replicates towards the ODNs used in this study between BSA, APE1 WT , APE1 NΔ33 , normalized on the respective total protein staining signal (Figure S4C). H) Graph representing the summary of APE1 WT and APE1 NΔ33 kinetic parameters, including kon and koff, towards each of the ODNs examined in MES buffer pH 5.5. KD values, expressed in nM, are reported (n=3). I) Representative crosslinking analysis with APE1 WT and APE1 NΔ33 recombinant proteins and each damaged iM (25 nM). On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Fluorescence, Negative Control, Marker, Binding Assay, Staining, Recombinant

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) Representative denaturing polyacrylamide gels of time-course kinetics APE1 WT cleavage activity. Lanes 1 and 2 indicate DNA fragments with known length (16 and 13 nucleotides, respectively). The “free” sample represents the control without protein (lane 3). On the right, the substrate and the product bands are indicated by two arrows and the expected length of the cleavage product is indicated upon the AP-site. A constant dose of APE1 (80 nM) was incubated with the respective oligonucleotide at 37°C. B) Relative graph depicting the time (expressed in minutes) and percentage of cleavage (%) on the x- and y-axis, respectively. Data are expressed as mean ± SD of three independent technical replicates. C) Representative denaturing polyacrylamide gels of APE1 WT (lane 2-5) and APE1 NΔ33 (lane 6-9) cleavage activity performed on all substrates (lane 2-9). “free” sample represents the control without protein (lane 1). On the right, the substrate and the product bands are indicated by two arrows. A constant dose of APE1 WT or APE1 NΔ33 (120 nM) was incubated with the respective oligonucleotide at 37°C, and the reactions were stopped at different time points, indicated upon the gel. D-G) Relative graphs illustrating the time-course kinetics activity of APE1 WT and APE1 NΔ33 recombinant proteins on AP14 (D), AP16 (E), AP17 (F) and AP20 (G). Time (expressed in minutes) and percentage of cleavage (%) are reported on the x- and y-axis, respectively. Data are expressed as mean ± SD of three independent technical replicates.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Activity Assay, Control, Incubation, Recombinant

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) PLA analysis of the interaction between APE1 and PCBP1 proteins in HeLa and U2OS cells (PLA dots in red). The merged panel shows the overlay between the four channels, including APE1 and PCBP1 staining in green (rabbit-488) and magenta (mouse-633), respectively, and the nuclear DAPI staining in blue. The scale is indicated and expressed in μm. B) Graph depicting the number of PLA dots per nucleus in U2OS and HeLa cell lines. Average and standard deviation values are plotted (n=3). C) Representative denaturing polyacrylamide gels of APE1 WT cleavage activity performed on all substrates (lane 3, 5-7), modulated by the co-incubation with PCBP1 (lane 5-7). Lane 2 is a control without any proteins. Lane 4 is a control with only the substrate and PCBP1. Lane 1 indicates a DNA fragment with a known length (16 nt). On the right, the substrate and the product bands are indicated by two arrows. The ODNs were pre-incubated with increasing amounts of PCBP1 (indicated upon the gel and expressed in nM) for 90 minutes at 4°C. A constant dose of APE1 WT (80 nM) was then added to the reactions and incubated at 37°C for 60 minutes. D) Relative graph illustrating the activity of APE1 WT recombinant protein after pre-incubation with different amounts of PCBP1 on AP14, AP16, AP17 and AP20. PCBP1 concentration (nM) and percentage of cleavage (%) are reported on the x- and y-axis, respectively. Data are expressed as mean ± SD of three independent technical replicates. E) Representative SWB shows the binding between the telomeric iM ODNs (reported upon the gel) and recombinant APE1 WT and PCBP1 proteins. BSA was used as a negative control. Each protein was loaded on the SDS-PAGE gel, blotted on the membrane and then incubated with the respective fluorescent probe (5 pmol). On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa. F) Relative histogram summarizing the different binding abilities towards each ODN between BSA, APE1 WT , PCBP1 (n=3), normalized on the respective total protein staining signal (Figure S6G).

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Staining, Standard Deviation, Activity Assay, Incubation, Control, Recombinant, Concentration Assay, Binding Assay, Negative Control, SDS Page, Membrane, Marker

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) TRF assay was used to measure telomere length in U2OS cells expressing (U2OS WT ) or knocked-out for APE1 (U2OS 19 ) protein, either silenced or not for PCBP1 protein (siPCBP1 and siSCR, respectively), as indicated upon the gel. On the left side of the gel, the molecular weight marker, as provided by the kit, is loaded, and the length of each band is indicated and expressed as bp. On the right side, a representative graph showing the intensity profiles relative to the TRF assay is reported. On the y-axis, the signal intensity is reported (expressed in a.u.), while on the x-axis the TRF marker length (expressed in bp) is reported. B) Graph reporting the normalized TRF mean length of U2OS WT and U2OS 19 . TRF mean length is normalized to U2OS WT values. Data are expressed as mean± SD of three independent replicates. C) Graph reporting the normalized TRF mean length of U2OS WT and U2OS 19 silenced for PCBP1. TRF mean length is normalized to each respective scramble silencing values. Data are expressed as mean± SD of three independent replicates. D) PLA analysis between TRF2 and APE1 proteins in U2OS cells, either silenced or not for PCBP1 protein (siPCBP1 and mock, siSCR, respectively). The representative merge panel shows PLA dots in red, TRF2 staining in green and APE1 staining in magenta. The nuclear staining, obtained with DAPI, is in blue. The scale is indicated and expressed in μm. E) Relative graph depicting the number of TRF2-APE1 PLA dots per nucleus in all U2OS WT conditions. Average and standard deviation values are plotted (n = 3). F) PLA analysis of the interaction between TRF2 and PCBP1 proteins in U2OS cells expressing (U2OS WT ) or knocked-out for APE1. The representative merge panel shows PLA dots in red, TRF2 staining in green and and PCBP1 in magenta. The nuclear staining, obtained with DAPI, is in blue. The scale is indicated and expressed in μm. G) Relative graph depicting the number of TRF2-PCBP1 PLA dots per nucleus in U2OS WT , U2OS 19 and U2OS 21 cell lines. Average and standard deviation values are plotted (n = 3). H) Representative immunofluorescence images of iMab antibody staining in U2OS WT , U2OS 19 and U2OS 21 cell lines. iMab foci are shown in green (rabbit 488 secondary antibody), while nuclei were stained with DAPI. The scale is indicated and expressed in μm. I) Scatter plot of iMab analysis representing individual values of iM foci in the nuclear compartment of U2OS clones. Foci were counted for each cell, using DAPI as a nuclear mask. Data are expressed as means ± SD of two independent replicates. J) Western blot analysis of p21, 𝛾H2AX, PCBP1 and APE1 levels in U2OS WT , U2OS 19 and U2OS 21 cell lines, either silenced or not for PCBP1 (siPCBP1 and siSCR, respectively). Tubulin was used as normalizer. Molecular weights (MW, expressed in kDa) are reported on the left. K-L) Densitometric analysis of 𝛾H2AX expression levels, normalized to tubulin. In the upper panel (K), fold change values relative to U2OS WT , arbitrarily set to 1, are shown. In the lower panel (L), fold change values relative to each siSCR, arbitrarily set to 1, are shown. Values are mean ± SD of four independent replicates. M-N) Densitometric analysis of p21 expression levels, normalized to tubulin. In the upper panel (M), fold change values relative to U2OS WT , arbitrarily set to 1, are shown. In the lower panel (N), fold change values relative to each siSCR, arbitrarily set to 1, are shown. Values are mean ± SD of four independent replicates.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: TRF Assay, Expressing, Molecular Weight, Marker, Staining, Standard Deviation, Immunofluorescence, Clone Assay, Western Blot

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) Representative SWB assay between the C-NAT and recombinant APE1 WT and APE1 NΔ33 proteins. BSA was used as a negative control. On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa. B) Relative histogram summarizing the different binding abilities of three independent replicates towards C-NAT between BSA, APE1 WT , APE1 NΔ33 , normalized on the respective total protein staining signal (Figure S2A). C) Graph representing the summary of C-NAT-APE1 WT and C-NAT-APE1 NΔ33 kinetic parameters (kon and koff), expressed in nM, in MES buffer pH 5.5. KD values, expressed in nM, are reported (n=3). D) Representation of 2D { 15 N- 1 H} TROESY-HSQC NMR spectra recorded at 25 °C. Measurements were performed with APE1 WT alone (red), 0.6 (green) and 1:1 (blue) molar ratios of C-NAT to APE1 WT . E) Depiction of NMR-derived binding analysis on APE1 structure (PDB code: 1bix). Cyan represents deposited canonical duplex DNA (for reference only). Purple represent residues which chemical shift variations (Δ8) that, before and after inclusion of i-motif DNA, are equal or superior to 2 standard deviations from the mean. Orange represent residues that lost over 2/3 or the respective peak volume. F) Schematic representation of the three consecutive telomeric iM (3x-C-NAT) embedded in a DNA sequence used for the single molecule analysis. G) Representative kymograph of APE1 WT -mEGFP from A549 clone 31 NCE binding to the iM (3x-C-NAT) substrate. Time (expressed in s) and position (expressed in µm) are reported on the x- and y-axis, respectively. The iM position is indicated with the red arrow. H) Cumulative residence time distribution (CRTD) plot of specific binding events of APE1 WT -mEGFP to 3x-C-NAT. I) Cumulative gap time distribution (CGTD) plot of APE1 WT -mEGFP association with 3x-C-NAT under the tested conditions.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Recombinant, Negative Control, Marker, Binding Assay, Staining, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) Scheme of the damaged telomeric iM ODNs, holding an abasic site at different positions (AP14, AP16, AP17, AP20), indicated by a star. B) CD spectrum of damaged ODNs. On the x-axis, the wavelength is reported (nm), while on the y-axis the CD value (mdeg). C) CD melting profile of damaged ODNs. Temperature (expressed in °C) and CD (expressed in mdeg) are reported on the x-and y-axis, respectively. D) Graph representing the summary of iMab kinetic parameters, including Kon and Koff, towards each of the ODNs examined in MES buffer pH 5.5. KD values, expressed in nM, are reported (n=3). E) Histogram showing fluorescence change of the different ODNs dye environment (expressed in %, y-axis) caused by increasing pH (x-axis), calculated in the plateau phase of five different replicates. F) Representative SWB assays between each ODNs, as indicated upon each panel, and APE1 WT or APE1 NΔ33 proteins. BSA was used as a negative control. On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa. G) Relative histogram summarizing the different binding abilities of three independent replicates towards the ODNs used in this study between BSA, APE1 WT , APE1 NΔ33 , normalized on the respective total protein staining signal (Figure S4C). H) Graph representing the summary of APE1 WT and APE1 NΔ33 kinetic parameters, including kon and koff, towards each of the ODNs examined in MES buffer pH 5.5. KD values, expressed in nM, are reported (n=3). I) Representative crosslinking analysis with APE1 WT and APE1 NΔ33 recombinant proteins and each damaged iM (25 nM). On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Fluorescence, Negative Control, Marker, Binding Assay, Staining, Recombinant

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) Representative denaturing polyacrylamide gels of time-course kinetics APE1 WT cleavage activity. Lanes 1 and 2 indicate DNA fragments with known length (16 and 13 nucleotides, respectively). The “free” sample represents the control without protein (lane 3). On the right, the substrate and the product bands are indicated by two arrows and the expected length of the cleavage product is indicated upon the AP-site. A constant dose of APE1 (80 nM) was incubated with the respective oligonucleotide at 37°C. B) Relative graph depicting the time (expressed in minutes) and percentage of cleavage (%) on the x- and y-axis, respectively. Data are expressed as mean ± SD of three independent technical replicates. C) Representative denaturing polyacrylamide gels of APE1 WT (lane 2-5) and APE1 NΔ33 (lane 6-9) cleavage activity performed on all substrates (lane 2-9). “free” sample represents the control without protein (lane 1). On the right, the substrate and the product bands are indicated by two arrows. A constant dose of APE1 WT or APE1 NΔ33 (120 nM) was incubated with the respective oligonucleotide at 37°C, and the reactions were stopped at different time points, indicated upon the gel. D-G) Relative graphs illustrating the time-course kinetics activity of APE1 WT and APE1 NΔ33 recombinant proteins on AP14 (D), AP16 (E), AP17 (F) and AP20 (G). Time (expressed in minutes) and percentage of cleavage (%) are reported on the x- and y-axis, respectively. Data are expressed as mean ± SD of three independent technical replicates.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Activity Assay, Control, Incubation, Recombinant

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) PLA analysis of the interaction between APE1 and PCBP1 proteins in HeLa and U2OS cells (PLA dots in red). The merged panel shows the overlay between the four channels, including APE1 and PCBP1 staining in green (rabbit-488) and magenta (mouse-633), respectively, and the nuclear DAPI staining in blue. The scale is indicated and expressed in μm. B) Graph depicting the number of PLA dots per nucleus in U2OS and HeLa cell lines. Average and standard deviation values are plotted (n=3). C) Representative denaturing polyacrylamide gels of APE1 WT cleavage activity performed on all substrates (lane 3, 5-7), modulated by the co-incubation with PCBP1 (lane 5-7). Lane 2 is a control without any proteins. Lane 4 is a control with only the substrate and PCBP1. Lane 1 indicates a DNA fragment with a known length (16 nt). On the right, the substrate and the product bands are indicated by two arrows. The ODNs were pre-incubated with increasing amounts of PCBP1 (indicated upon the gel and expressed in nM) for 90 minutes at 4°C. A constant dose of APE1 WT (80 nM) was then added to the reactions and incubated at 37°C for 60 minutes. D) Relative graph illustrating the activity of APE1 WT recombinant protein after pre-incubation with different amounts of PCBP1 on AP14, AP16, AP17 and AP20. PCBP1 concentration (nM) and percentage of cleavage (%) are reported on the x- and y-axis, respectively. Data are expressed as mean ± SD of three independent technical replicates. E) Representative SWB shows the binding between the telomeric iM ODNs (reported upon the gel) and recombinant APE1 WT and PCBP1 proteins. BSA was used as a negative control. Each protein was loaded on the SDS-PAGE gel, blotted on the membrane and then incubated with the respective fluorescent probe (5 pmol). On the left, the electrophoretic marker is loaded, and the different molecular weights are expressed in kDa. F) Relative histogram summarizing the different binding abilities towards each ODN between BSA, APE1 WT , PCBP1 (n=3), normalized on the respective total protein staining signal (Figure S6G).

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: Staining, Standard Deviation, Activity Assay, Incubation, Control, Recombinant, Concentration Assay, Binding Assay, Negative Control, SDS Page, Membrane, Marker

Journal: bioRxiv

Article Title: Apurinic/apyrimidinic endodeoxyribonuclease 1 contributes to the repair of damaged intercalated-motif of telomeric sequences

doi: 10.64898/2025.12.17.694817

Figure Lengend Snippet: A) TRF assay was used to measure telomere length in U2OS cells expressing (U2OS WT ) or knocked-out for APE1 (U2OS 19 ) protein, either silenced or not for PCBP1 protein (siPCBP1 and siSCR, respectively), as indicated upon the gel. On the left side of the gel, the molecular weight marker, as provided by the kit, is loaded, and the length of each band is indicated and expressed as bp. On the right side, a representative graph showing the intensity profiles relative to the TRF assay is reported. On the y-axis, the signal intensity is reported (expressed in a.u.), while on the x-axis the TRF marker length (expressed in bp) is reported. B) Graph reporting the normalized TRF mean length of U2OS WT and U2OS 19 . TRF mean length is normalized to U2OS WT values. Data are expressed as mean± SD of three independent replicates. C) Graph reporting the normalized TRF mean length of U2OS WT and U2OS 19 silenced for PCBP1. TRF mean length is normalized to each respective scramble silencing values. Data are expressed as mean± SD of three independent replicates. D) PLA analysis between TRF2 and APE1 proteins in U2OS cells, either silenced or not for PCBP1 protein (siPCBP1 and mock, siSCR, respectively). The representative merge panel shows PLA dots in red, TRF2 staining in green and APE1 staining in magenta. The nuclear staining, obtained with DAPI, is in blue. The scale is indicated and expressed in μm. E) Relative graph depicting the number of TRF2-APE1 PLA dots per nucleus in all U2OS WT conditions. Average and standard deviation values are plotted (n = 3). F) PLA analysis of the interaction between TRF2 and PCBP1 proteins in U2OS cells expressing (U2OS WT ) or knocked-out for APE1. The representative merge panel shows PLA dots in red, TRF2 staining in green and and PCBP1 in magenta. The nuclear staining, obtained with DAPI, is in blue. The scale is indicated and expressed in μm. G) Relative graph depicting the number of TRF2-PCBP1 PLA dots per nucleus in U2OS WT , U2OS 19 and U2OS 21 cell lines. Average and standard deviation values are plotted (n = 3). H) Representative immunofluorescence images of iMab antibody staining in U2OS WT , U2OS 19 and U2OS 21 cell lines. iMab foci are shown in green (rabbit 488 secondary antibody), while nuclei were stained with DAPI. The scale is indicated and expressed in μm. I) Scatter plot of iMab analysis representing individual values of iM foci in the nuclear compartment of U2OS clones. Foci were counted for each cell, using DAPI as a nuclear mask. Data are expressed as means ± SD of two independent replicates. J) Western blot analysis of p21, 𝛾H2AX, PCBP1 and APE1 levels in U2OS WT , U2OS 19 and U2OS 21 cell lines, either silenced or not for PCBP1 (siPCBP1 and siSCR, respectively). Tubulin was used as normalizer. Molecular weights (MW, expressed in kDa) are reported on the left. K-L) Densitometric analysis of 𝛾H2AX expression levels, normalized to tubulin. In the upper panel (K), fold change values relative to U2OS WT , arbitrarily set to 1, are shown. In the lower panel (L), fold change values relative to each siSCR, arbitrarily set to 1, are shown. Values are mean ± SD of four independent replicates. M-N) Densitometric analysis of p21 expression levels, normalized to tubulin. In the upper panel (M), fold change values relative to U2OS WT , arbitrarily set to 1, are shown. In the lower panel (N), fold change values relative to each siSCR, arbitrarily set to 1, are shown. Values are mean ± SD of four independent replicates.

Article Snippet: E. coli BL21 (DE3) bacteria (C2530H, New England Biolabs) were transformed with 100 ng of plasmids pGEX-3X APE1 WT , pGEX-3X APE1 NΔ33 , pTAC-MAT-APE1 WT -GFP, pTAC-MAT-APE1 NΔ33 -GFP or pET166-E1 for the expression of GST-tagged APE1 WT , APE1 NΔ33 , His- and GFP-tagged APE1 WT , APE1 NΔ33 and His-tagged PCBP1, respectively.

Techniques: TRF Assay, Expressing, Molecular Weight, Marker, Staining, Standard Deviation, Immunofluorescence, Clone Assay, Western Blot