Journal: bioRxiv

Article Title: TRF2 Non-Telomeric Function is Indispensable for Neural stemness

doi: 10.1101/2025.02.28.640739

Figure Lengend Snippet: a. Schematic of Terf2 F/F / Nes:Cre mice. b. Neurosphere assay with mNSCs from Terf2 f/f / Nes:Cre mice. 5 days post treatment with (4– OHT) tamoxifen and (EtOH) vehicle control. The graph indicates the number of neurospheres formed per well (error bar ± SD (unpaired t test)) c. Flow cytometry of mNSCs from Terf2 F/F / Nes:Cre mice; Vehicle control (EtOH) and TRF2 knockout cells (4-OHT). Mean intensity of fluorescence (MIF) for (i) Ki67, (ii) MAP2 (iii) BDNF, (iv) L1CAM and TRF2 (supplementary) is shown. The cell counts are normalized to respective modes for comparative representation. d. Immunofluorescence staining of TRF2 and β-III Tubulin proteins in normal and TRF2 silenced SH-SY5Y cells. β- III Tubulin and TRF2 were stained using Alexa fluor-594 (red signal) and Alexa fluor-488 (green signal), respectively. The graph shows the quantification of β- III Tubulin positive neurite from 30 cells (n = 30) shown in respective right panels (error bars ± SE). e. Flow cytometry of Control and TRF2 silenced SH-SY5Y cells. Mean intensity of fluorescence (MIF) for (i) Ki67 (ii) MAP2, (iii) BDNF, (iv) L1CAM and TRF2 (supplementary) is shown. The cell counts are normalized to respective modes for comparative representation. f. Immunofluorescence staining for TRF2 and β- III Tubulin proteins in Control, WT TRF2 and TRF2 ΔBΔM overexpression conditions. β- III Tubulin and TRF2 were stained using Alexa fluor-594 (red signal) and Alexa fluor-498 (green signal), respectively. Quantification of β- III Tubulin positive neurite from 30 cells (n = 30) shown in respective right panels (error bars ± SE). g. Flow cytometry using staining for MAP2 and TRF2 in SH-SY5Y cells. Control, WT TRF2 and TRF2 ΔBΔM overexpression conditions. Mean intensity of fluorescence (MIF) for MAP2 and TRF2 is shown (supplementary). The cell counts are normalized to respective modes for comparative representation

Article Snippet: Primary antibodies (Ki67 abcam#ab15580, γH2AX CST #S139 (20E3), 53BP1 abcam #ab36823, p-ATM CST #S1981(D25E5), MAP2 abcam #ab32454, BDNF abcam #ab108319, L1CAM abcam #ab208155, and TRF2 Santa Cruz #sc52968/ Merck #4A794)were diluted in 1% BSA (in PBS) at a 1:250 volume ratio and incubated with the cells as a cocktail for 2 hours at room temperature.

Techniques: Neurosphere Assay, Control, Flow Cytometry, Knock-Out, Fluorescence, Immunofluorescence, Staining, Over Expression

Journal: bioRxiv

Article Title: TRF2 Non-Telomeric Function is Indispensable for Neural stemness

doi: 10.1101/2025.02.28.640739

Figure Lengend Snippet: a. Flow cytometry with staining for γH2AX (i), 53BP1 (ii), p-ATM (iii) in mNSCs of Terf2 F/F / Nes:Cre mice. Vehicle control (EtOH) and TRF2 knockout cells (4-OHT). Mean intensity of fluorescence is shown. The cell counts are normalized to respective modes for comparative representation b. Flow cytometry with staining for γH2AX (i), 53BP1 (ii), p-ATM (iii) in SH-SY5Y cells. Control and TRF2 silenced cells. Mean intensity of fluorescence (MIF) is shown. The cell counts are normalized to respective modes for comparative representation. c. Flow cytometry with staining for γH2AX (i), 53BP1 (ii), p-ATM (iii) in SH-SY5Y cells. Control and TRF2 ΔBΔM overexpression cells. Mean intensity of fluorescence (MIF) is shown. The cell counts are normalized to respective modes for comparative representation. d. Representative immunofluorescence and fluorescence in situ hybridization (IF–FISH) for 53BP1 (red) and telomeres (green) inSHSY5Y cells and Fibroblasts treated as indicated. Scale bar, (3 µm). e, Quantification of the percentage of cells with more than 10 53BP1 foci colocalizing with telomeres e. Quantification of the percentage of cells with more than 10 53BP1 foci colocalizing with telomeres, detected as in d. Error bars indicate standard deviation

Article Snippet: Primary antibodies (Ki67 abcam#ab15580, γH2AX CST #S139 (20E3), 53BP1 abcam #ab36823, p-ATM CST #S1981(D25E5), MAP2 abcam #ab32454, BDNF abcam #ab108319, L1CAM abcam #ab208155, and TRF2 Santa Cruz #sc52968/ Merck #4A794)were diluted in 1% BSA (in PBS) at a 1:250 volume ratio and incubated with the cells as a cocktail for 2 hours at room temperature.

Techniques: Flow Cytometry, Staining, Control, Knock-Out, Fluorescence, Over Expression, Immunofluorescence, In Situ Hybridization, Standard Deviation

Journal: bioRxiv

Article Title: TRF2 Non-Telomeric Function is Indispensable for Neural stemness

doi: 10.1101/2025.02.28.640739

Figure Lengend Snippet: a. TRF2 ChIP followed by differentiation genes promoter qPCR for TRF2 binding in mNSCs (a) and SH-SY5Y (b) c. Gene expression changes upon tamoxifen (4-OHT) treated mNSC after 5 days. Fold change normalized over cells treated with vehicle control EtOH. d. Effect of TRF2 silencing on differentiation gene expression at increasing time points post transfection. Fold change normalized over cells treated with scrambled siRNA control. e. Pol2 (Ser5) occupancy on the promoters of differentiation genes following TRF2 silencing in (mNSCs (e) and SHSY5Y (f)). g. Gene expression changes upon DNA binding mutant expression of TRF2 in mNSC (g) and SH-SY5Y(h).

Article Snippet: Primary antibodies (Ki67 abcam#ab15580, γH2AX CST #S139 (20E3), 53BP1 abcam #ab36823, p-ATM CST #S1981(D25E5), MAP2 abcam #ab32454, BDNF abcam #ab108319, L1CAM abcam #ab208155, and TRF2 Santa Cruz #sc52968/ Merck #4A794)were diluted in 1% BSA (in PBS) at a 1:250 volume ratio and incubated with the cells as a cocktail for 2 hours at room temperature.

Techniques: Binding Assay, Gene Expression, Control, Transfection, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: TRF2 Non-Telomeric Function is Indispensable for Neural stemness

doi: 10.1101/2025.02.28.640739

Figure Lengend Snippet: a. Effect of TRF2 silencing on H3K27me3 occupancy on the promoters of differentiation genes in mNSC (a) and SH-SY5Y (b). c. EZH2 occupancy on the promoters of differentiation genes on silencing TRF2 in mNSC (c) and SH-SY5Y (d). e. REST occupancy on the promoters of differentiation genes upon TRF2 silencing mNSC (e) and SH-SY5Y (f).

Article Snippet: Primary antibodies (Ki67 abcam#ab15580, γH2AX CST #S139 (20E3), 53BP1 abcam #ab36823, p-ATM CST #S1981(D25E5), MAP2 abcam #ab32454, BDNF abcam #ab108319, L1CAM abcam #ab208155, and TRF2 Santa Cruz #sc52968/ Merck #4A794)were diluted in 1% BSA (in PBS) at a 1:250 volume ratio and incubated with the cells as a cocktail for 2 hours at room temperature.

Techniques:

Journal: bioRxiv

Article Title: TRF2 Non-Telomeric Function is Indispensable for Neural stemness

doi: 10.1101/2025.02.28.640739

Figure Lengend Snippet: a. Immunofluorescence staining for TRF2 and β- III Tubulin proteins in Control and TRF2 K176R expression conditions. β- III Tubulin and TRF2 were stained using Alexa fluor-594 (red signal) and Alexa fluor-488 (green signal), respectively. Quantification of β- III Tubulin positive neurite from 30 cells (n = 30) shown in respective right panels (error bars ± SE). b. Effect of TRF2 K176R on differentiation gene expression 108 hrs post transfection. c. Co- Immunoprecipitation of WT TRF2 and K176R TRF2. d. REST occupancy on the promoters of differentiation genes in the presence of TRF2 K176R. e. Effect of TRF2 K176R on H3K27me3 occupancy on the promoters of differentiation genes. f. Occupancy of TRF2 K176R on the promoters of differentiation genes. ChIP for FLAG (TRF2).

Article Snippet: Primary antibodies (Ki67 abcam#ab15580, γH2AX CST #S139 (20E3), 53BP1 abcam #ab36823, p-ATM CST #S1981(D25E5), MAP2 abcam #ab32454, BDNF abcam #ab108319, L1CAM abcam #ab208155, and TRF2 Santa Cruz #sc52968/ Merck #4A794)were diluted in 1% BSA (in PBS) at a 1:250 volume ratio and incubated with the cells as a cocktail for 2 hours at room temperature.

Techniques: Immunofluorescence, Staining, Control, Expressing, Gene Expression, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: TRF2 Non-Telomeric Function is Indispensable for Neural stemness

doi: 10.1101/2025.02.28.640739

Figure Lengend Snippet: a. BG4 ChIP followed by q-PCR for the promoters of genes associated to differentiation in SH-SY5Y cells. b. Expression of genes negatively regulated by TRF2 upon DHX36 overexpression. c. Quantification of luciferase expression driven by APP, GFRA1, MAP2 and L1CAM promoters upon TRF2 silencing d. Quantification of luciferase expression driven by GFRA1, MAP2 promoters with the corresponding mutant G4. e. Quantification of luciferase expression driven by GFRA1, MAP2 promoters with DHX36 expression. f. FACS based quantification of MAP2 promoter driven GFP expression upon treatment of ligands at 1.5 uM concentration for 5 days. g. Expression of genes regulated by TRF2 upon treatment of ligands at 1.5 uM concentration for 108hrs. h. Immunofluorescence staining for β- III Tubulin proteins in Control and Ligand treatment conditions for 7 days. β- III Tubulin was stained using Alexa fluor-594 (red signal). Scale bar 200 µm.

Article Snippet: Primary antibodies (Ki67 abcam#ab15580, γH2AX CST #S139 (20E3), 53BP1 abcam #ab36823, p-ATM CST #S1981(D25E5), MAP2 abcam #ab32454, BDNF abcam #ab108319, L1CAM abcam #ab208155, and TRF2 Santa Cruz #sc52968/ Merck #4A794)were diluted in 1% BSA (in PBS) at a 1:250 volume ratio and incubated with the cells as a cocktail for 2 hours at room temperature.

Techniques: Expressing, Over Expression, Luciferase, Mutagenesis, Concentration Assay, Immunofluorescence, Staining, Control

Journal: Antioxidants

Article Title: Nicotinamide Riboside and Phycocyanin Oligopeptides Affect Stress Susceptibility in Chronic Corticosterone-Exposed Rats

doi: 10.3390/antiox12101849

Figure Lengend Snippet: Effects of NR and PC oligopeptides on liver POT1a ( A ), POT1b ( B ), TRF1 ( C ), TRF2 ( D ), and Tin2 ( E ) levels in rats subject to chronic corticosterone (CORT). The densitometric analysis of the relative intensity according to the control group of the Western blotting bands was performed with β-actin normalization to ensure equal protein loading ( F ). Blots were repeated at least three times ( n = 3), and a representative blot is shown. Data are expressed as a percent of the control set at 100%. The error bars above the lines indicate the standard deviation of the mean. Different symbols (a–f) indicate significant differences among the groups (ANOVA and Tukey post hoc test; p < 0.05). POT1a, protection of telomeres protein 1a; POT1b, protection of telomeres protein 1b; TRF1, telomeric repeat-binding factor 1; TRF2, telomeric repeat-binding factor 1; Tin2, TRF1-interacting protein 2. CS: chronic stress; NR: Nicotinamide riboside (26.44 mg/kg); PC-LD: Phycocyanin oligopeptide, low dose (2.64 mg/kg); PC-HD: Phycocyanin oligopeptide, high dose (26.44 mg/kg). Rats except those of the control group were given daily corticosterone injections (40 mg/kg) to induce stress conditions, or NR and PC were orally administered for 21 days. Full immunoblots presented in .

Article Snippet: NC membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with rat-specific primary antibodies diluted to 1:1000 (IL-6 (sc-57315), TNF-α (sc-52746), IL-1β (sc-515598), TRF2 (sc-47693), Tin2 (sc-73177), SIRT3 (sc-365175), NAMPT (sc-393444) (Santa Cruz Biotechnology, Heidelberg, Germany), IL-8 (NBP2-16958), POTlb (NBP2-50255) (Novus Biotech, CO, USA), POT1a (PA5-75366), SIRT1 (MA5-27217), and TRF1 (MA1-46375) (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C.

Techniques: Control, Western Blot, Standard Deviation, Binding Assay

Journal: Antioxidants

Article Title: Nicotinamide Riboside and Phycocyanin Oligopeptides Affect Stress Susceptibility in Chronic Corticosterone-Exposed Rats

doi: 10.3390/antiox12101849

Figure Lengend Snippet: The experimental design. CS: chronic stress induced with corticosterone; NR: nicotinamide riboside (26.44 mg/kg); PC-LD: Phycocyanin oligopeptide, low dose (2.64 mg/kg); PC-HD: Phycocyanin oligopeptide, high dose (26.44 mg/kg). Rats except those of the control group were given daily corticosterone injections (40 mg/kg) to induce stress conditions or NR and PC were orally administered for 21 days.

Article Snippet: NC membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with rat-specific primary antibodies diluted to 1:1000 (IL-6 (sc-57315), TNF-α (sc-52746), IL-1β (sc-515598), TRF2 (sc-47693), Tin2 (sc-73177), SIRT3 (sc-365175), NAMPT (sc-393444) (Santa Cruz Biotechnology, Heidelberg, Germany), IL-8 (NBP2-16958), POTlb (NBP2-50255) (Novus Biotech, CO, USA), POT1a (PA5-75366), SIRT1 (MA5-27217), and TRF1 (MA1-46375) (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C.

Techniques: Control

Journal: Antioxidants

Article Title: Nicotinamide Riboside and Phycocyanin Oligopeptides Affect Stress Susceptibility in Chronic Corticosterone-Exposed Rats

doi: 10.3390/antiox12101849

Figure Lengend Snippet: Effects of NR and PC oligopeptides on liver IL-6 ( A ), TNF-α ( B ), IL-1β ( C ), and IL-8 ( D ) levels in rats subject to chronic corticosterone (CORT). The densitometric analysis of the relative intensity according to the control group of the Western blotting bands was performed with β-actin normalization to ensure equal protein loading ( E ). Blots were repeated at least three times ( n = 3), and a representative blot is shown. Data are expressed as a percent of the control set at 100%. The error bars above the lines indicate the standard deviation of the mean. Different symbols (a–f) indicate significant differences among the groups (ANOVA and Tukey post hoc test; p < 0.05). IL-6, interleukin-6; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; IL-8, interleukin-8. CS: chronic stress; NR: Nicotinamide riboside (26.44 mg/kg); PC-LD: Phycocyanin oligopeptide, low dose (2.64 mg/kg); PC-HD: Phycocyanin oligopeptide, high dose (26.44 mg/kg). Rats except those of the control group were given daily corticosterone injections (40 mg/kg) to induce stress conditions, or NR and PC were orally administered for 21 days. Full immunoblots presented in .

Article Snippet: NC membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with rat-specific primary antibodies diluted to 1:1000 (IL-6 (sc-57315), TNF-α (sc-52746), IL-1β (sc-515598), TRF2 (sc-47693), Tin2 (sc-73177), SIRT3 (sc-365175), NAMPT (sc-393444) (Santa Cruz Biotechnology, Heidelberg, Germany), IL-8 (NBP2-16958), POTlb (NBP2-50255) (Novus Biotech, CO, USA), POT1a (PA5-75366), SIRT1 (MA5-27217), and TRF1 (MA1-46375) (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C.

Techniques: Control, Western Blot, Standard Deviation

Journal: Antioxidants

Article Title: Nicotinamide Riboside and Phycocyanin Oligopeptides Affect Stress Susceptibility in Chronic Corticosterone-Exposed Rats

doi: 10.3390/antiox12101849

Figure Lengend Snippet: Effects of NR and PC oligopeptides on liver POT1a ( A ), POT1b ( B ), TRF1 ( C ), TRF2 ( D ), and Tin2 ( E ) levels in rats subject to chronic corticosterone (CORT). The densitometric analysis of the relative intensity according to the control group of the Western blotting bands was performed with β-actin normalization to ensure equal protein loading ( F ). Blots were repeated at least three times ( n = 3), and a representative blot is shown. Data are expressed as a percent of the control set at 100%. The error bars above the lines indicate the standard deviation of the mean. Different symbols (a–f) indicate significant differences among the groups (ANOVA and Tukey post hoc test; p < 0.05). POT1a, protection of telomeres protein 1a; POT1b, protection of telomeres protein 1b; TRF1, telomeric repeat-binding factor 1; TRF2, telomeric repeat-binding factor 1; Tin2, TRF1-interacting protein 2. CS: chronic stress; NR: Nicotinamide riboside (26.44 mg/kg); PC-LD: Phycocyanin oligopeptide, low dose (2.64 mg/kg); PC-HD: Phycocyanin oligopeptide, high dose (26.44 mg/kg). Rats except those of the control group were given daily corticosterone injections (40 mg/kg) to induce stress conditions, or NR and PC were orally administered for 21 days. Full immunoblots presented in .

Article Snippet: NC membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with rat-specific primary antibodies diluted to 1:1000 (IL-6 (sc-57315), TNF-α (sc-52746), IL-1β (sc-515598), TRF2 (sc-47693), Tin2 (sc-73177), SIRT3 (sc-365175), NAMPT (sc-393444) (Santa Cruz Biotechnology, Heidelberg, Germany), IL-8 (NBP2-16958), POTlb (NBP2-50255) (Novus Biotech, CO, USA), POT1a (PA5-75366), SIRT1 (MA5-27217), and TRF1 (MA1-46375) (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C.

Techniques: Control, Western Blot, Standard Deviation, Binding Assay

Journal: Antioxidants

Article Title: Nicotinamide Riboside and Phycocyanin Oligopeptides Affect Stress Susceptibility in Chronic Corticosterone-Exposed Rats

doi: 10.3390/antiox12101849

Figure Lengend Snippet: Effects of NR and PC oligopeptides on liver SIRT1 ( A ), SIRT3 ( B ), and NAMPT ( C ) levels in rats subject to chronic corticosterone (CORT). The densitometric analysis of the relative intensity according to the control group of the Western blotting bands was performed with β-actin normalization to ensure equal protein loading ( D ). Blots were repeated at least three times ( n = 3), and a representative blot is shown. Data are expressed as a percent of the control set at 100%. The error bars above the lines indicate the standard deviation of the mean. Different symbols (a–e) indicate significant differences among the groups (ANOVA and Tukey post hoc test; p < 0.05). SIRT1, sirtuin 1; SIRT3, sirtuin 3; NAMPT, nicotinamide phosphoribosyltransferase. CS: chronic stress; NR: Nicotinamide riboside (26.44 mg/kg); PC-LD: Phycocyanin oligopeptide, low dose (2.64 mg/kg); PC-HD: Phycocyanin oligopeptide, high dose (26.44 mg/kg). Rats except those of the control group were given daily corticosterone injections (40 mg/kg) to induce stress conditions, or NR and PC were orally administered for 21 days. Full immunoblots presented in .

Article Snippet: NC membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with rat-specific primary antibodies diluted to 1:1000 (IL-6 (sc-57315), TNF-α (sc-52746), IL-1β (sc-515598), TRF2 (sc-47693), Tin2 (sc-73177), SIRT3 (sc-365175), NAMPT (sc-393444) (Santa Cruz Biotechnology, Heidelberg, Germany), IL-8 (NBP2-16958), POTlb (NBP2-50255) (Novus Biotech, CO, USA), POT1a (PA5-75366), SIRT1 (MA5-27217), and TRF1 (MA1-46375) (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 °C.

Techniques: Control, Western Blot, Standard Deviation