Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: The aberrant gene expression profile in IDD patients. (A) The heat maps of the consistently downregulated and upregulated genes in different IVD degeneration patients. The mRNAs from IDD patients who underwent different Pfirrmann grades (from 0 to 5) were subjected to microarray analysis. The heat maps indicated high (red) or low (green) levels of gene expression. (B-G) Verification of mRNA levels in IDD patients. The qRT-PCR was performed to verify the expression of three downregulated genes, including PolE (B), PolE2 (C), and PolE3 (D), and three upregulated genes, including Caspase-3 (E), Caspase-8 (F), and DDX59 (G). *P < 0.05, **P < 0.001.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: Iron supplementation restores PolE protein level and decreases cell apoptosis. The primary NP cells from IDD patients who underwent different Pfirrmann grades (0, 2 and 4) were treated with (+) or without (-) 100 μM FeCl3. After 24 hr, cells were collected and lysed, followed by immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control. F represents full length; C represents cleavage.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Western Blot

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: Iron depletion decreases PolE protein level and causes apoptosis. The human NP cells were treated with different concentrations (0, 20, 40 and 60 µM) of iron chelator DFO. After 24 hr, half of the DFO-treated cells were collected and lysed, followed by immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control (A). The other half of DFO-treated cells were collected and stained with Annexin V-PE/7-AAD, followed by flow cytometry analysis (B-E). (B) No DFO treatment; (C) 20 µM; (D) 40 µM; and (E) 60 µM DFO treatment.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Western Blot, Staining, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: PolE knockdown induces apoptosis. The human NP cells were infected with lentiviruses containing either control shRNA or three PolE-specific shRNAs. After selection in puromycin-containing medium, the control shRNA transfected cells (control) and PolE knockdown cells (KD-1, -2 and -3) were subjected to immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control (A). The same cells used in (A) were also collected and stained with Annexin V-PE/7-AAD, followed by flow cytometry analysis (B-E). (B) Control cells; (C) KD-1 cells; (D) KD-2 cells; and (E) KD-3 cells.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Infection, shRNA, Selection, Transfection, Western Blot, Staining, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: Overexpression of PolE in DMT1 or TfR1 knockdown cells is unable to reduce apoptosis. The DMT1-KD1 and TfR1-KD1 cells were transfected with pCDNA3-2xFlag or pCDNA3-PolE-2xFlag plasmid, respectively, to obtain PolE overexpression cell lines (DMT1-KD1+PolE and TfR1-KD1+PolE). Then, these two cell lines, together with NP, DMT1-KD1 and TfR1-KD1 cells were subjected to immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control (A). The same cells used in (A) were also collected and stained with Annexin V-PE/7-AAD, followed by flow cytometry analysis (B-F). (B) NP cells; (C) DMT1-KD1 cells; (D) DMT1-KD1+PolE cells; (E) TfR1-KD1 cells; and (F) TfR1-KD1+PolE cells.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Staining, Flow Cytometry

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet: POLE2 is highly expressed in ESCC tissues and the construction of POLE2 knockdown cell model. a Expression levels of POLE2 in ESCC tumor tissues and normal skin tissues were detected by IHC staining. b POLE2 expression and overall survival of ESCC by Kaplan–Meier survival analysis. c Transfection efficiency for Eca-109 and TE-1 cells was evaluated by expression of green fluorescent protein 72 h post-infection. d , e The specificity and validity of the lentivirus-mediated shRNA knockdown of POLE2 expression was verified by qPCR ( d ) and western blot analysis ( e ). The data were presented as the mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques: Expressing, Immunohistochemistry, Transfection, Infection, shRNA, Western Blot

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet: Relationship between POLE2 expression and tumor characteristics in patients with esophagus cancer

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques: Expressing

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet: Relationship between POLE2 expression and tumor characteristics in patients with esophagus cancer

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques: Expressing

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet: Knockdown of POLE2 inhibits cell proliferation, promotes apoptosis in ESCC cells. a Cell proliferation of Eca-109 and TE-1 cells with or without knockdown of POLE2 was evaluated by MTT assay. b Colony formation was evaluated for Eca-109 and TE-1 cells with or without POLE2 knockdown. c Flow cytometry analysis based on Annexin V-APC staining was utilized to detect the percentage of early apoptotic cell for Eca-109 and TE-1 cells. The X axis indicated the cell apoptosis while the Y axis indicated the green fluorescence detected from the GFP tagged on lentivirus (shPOLE2 or shCtrl). The data were expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques: MTT Assay, Flow Cytometry, Staining, Fluorescence

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet: Knockdown of POLE2 inhibits cell migration in ESCC cells. a Cell migration of Eca-109 and TE-1 cells with or without knockdown of POLE2 was evaluated by wound-healing assay. b Cell migration of Eca-109 and TE-1 cells with or without knockdown of POLE2 was evaluated by Transwell assay. The data were expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques: Migration, Wound Healing Assay, Transwell Assay

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet: Exploration of downstream molecular mechanism of POLE2 in ESCC cells. a Human apoptosis antibody array analysis was performed in Eca-109 cells with or without POLE2 knockdown. b Differences in human apoptotic antibody array were analyzed in Eca-109 cells regardless of POLE2 knockdown. c Densitometric analysis was performed and the gray values of differentially expressed proteins were shown. d The expression of pathway was observed by western blot in Eca-109 cells. The data were expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques: Ab Array, Expressing, Western Blot

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet:

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques: Sequencing

Journal: Cancer Cell International

Article Title: POLE2 knockdown reduce tumorigenesis in esophageal squamous cells

doi: 10.1186/s12935-020-01477-4

Figure Lengend Snippet:

Article Snippet: POLE2 , 59 , 1:1000 , Rabbit , Abcam , AB180214.

Techniques:

Journal: Journal of Cancer

Article Title: Prognostic and chemotherapeutic response prediction by proliferation essential gene signature: Investigating POLE2 in bladder cancer progression and cisplatin resistance

doi: 10.7150/jca.93023

Figure Lengend Snippet: POLE2 expression in the BLCA and its relationship with overall survival. (A&B) Random forest displaying the most important proliferation essential gene (PEG) — POLE2 among the 10 prognostic PEGs. (C-G) Comparisons of POLE2 expression between the BLCA and normal tissues from the datasets (C) GSE13507, (D) GSE37851, (E) GSE40335, (F) GSE52519 and (G) GSE65635. (H) Overall survival difference in BLCA patients with high- and low-POLE2 expression from the GSE13507 dataset. (I&J) Representative immunohistochemistry (IHC) images of POLE2 protein in (I) the BLCA tissues and (J) paired normal urothelial tissues. (K) Histogram of the IHC score revealing markedly higher protein expression of POLE2 in BLCA tissues than in normal tissues. (L) An enhanced protein level of POLE2 in the muscle-invasive BLCA compared to the non-muscle invasive BLCA suggested by the IHC assay. (M) Comparison of POLE2 protein levels between the high-grade non-muscle invasive BLCA and the low-grade non-muscle invasive using the IHC assay. (N) Comparison of POLE2 protein levels between the high-grade muscle invasive BLCA and the low-grade muscle invasive using the IHC assay. *, p <0.05; **, p <0.01; ***, p <0.001.

Article Snippet: Slides were incubated with anti-POLE2 antibodies (21146-1-AP, Proteintech, Wuhan, China, diluted 1:400) and then assessed by two pathologists.

Techniques: Expressing, Immunohistochemistry, Comparison

Journal: Journal of Cancer

Article Title: Prognostic and chemotherapeutic response prediction by proliferation essential gene signature: Investigating POLE2 in bladder cancer progression and cisplatin resistance

doi: 10.7150/jca.93023

Figure Lengend Snippet: The effects of POLE2 knockdown on the BLCA cell stemness, proliferation, invasion and migration. (A) The relationship between the POLE2 expression and RNA stemness score (RNAss) in the BLCA. (B) POLE2 expression in the T24 cells transfected with POLE2-short hairpin RNAs (POLE2-shRNAs, including POLE2-shRNA1 and POLE2-shRNA2). (C-F) Effects of POLE2 knockdown on (C) T24 cell stemness assessed by the clonogenic assay, (D) T24 cell growth determined by the CCK-8 assay, (E) T24 cell migration distance after 48 hours transfection determined by the scratch assay, and (F) the T24 cell invasion capability. (G) Effects of POLE2 knockdown on cisplatin chemotherapy resistance. **, p <0.01.

Article Snippet: Slides were incubated with anti-POLE2 antibodies (21146-1-AP, Proteintech, Wuhan, China, diluted 1:400) and then assessed by two pathologists.

Techniques: Knockdown, Migration, Expressing, Transfection, Clonogenic Assay, CCK-8 Assay, Wound Healing Assay

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: The aberrant gene expression profile in IDD patients. (A) The heat maps of the consistently downregulated and upregulated genes in different IVD degeneration patients. The mRNAs from IDD patients who underwent different Pfirrmann grades (from 0 to 5) were subjected to microarray analysis. The heat maps indicated high (red) or low (green) levels of gene expression. (B-G) Verification of mRNA levels in IDD patients. The qRT-PCR was performed to verify the expression of three downregulated genes, including PolE (B), PolE2 (C), and PolE3 (D), and three upregulated genes, including Caspase-3 (E), Caspase-8 (F), and DDX59 (G). *P < 0.05, **P < 0.001.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: Iron supplementation restores PolE protein level and decreases cell apoptosis. The primary NP cells from IDD patients who underwent different Pfirrmann grades (0, 2 and 4) were treated with (+) or without (-) 100 μM FeCl3. After 24 hr, cells were collected and lysed, followed by immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control. F represents full length; C represents cleavage.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Western Blot

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: Iron depletion decreases PolE protein level and causes apoptosis. The human NP cells were treated with different concentrations (0, 20, 40 and 60 µM) of iron chelator DFO. After 24 hr, half of the DFO-treated cells were collected and lysed, followed by immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control (A). The other half of DFO-treated cells were collected and stained with Annexin V-PE/7-AAD, followed by flow cytometry analysis (B-E). (B) No DFO treatment; (C) 20 µM; (D) 40 µM; and (E) 60 µM DFO treatment.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Western Blot, Staining, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: PolE knockdown induces apoptosis. The human NP cells were infected with lentiviruses containing either control shRNA or three PolE-specific shRNAs. After selection in puromycin-containing medium, the control shRNA transfected cells (control) and PolE knockdown cells (KD-1, -2 and -3) were subjected to immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control (A). The same cells used in (A) were also collected and stained with Annexin V-PE/7-AAD, followed by flow cytometry analysis (B-E). (B) Control cells; (C) KD-1 cells; (D) KD-2 cells; and (E) KD-3 cells.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Infection, shRNA, Selection, Transfection, Western Blot, Staining, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Iron deficiency accelerates intervertebral disc degeneration through affecting the stability of DNA polymerase epsilon complex

doi:

Figure Lengend Snippet: Overexpression of PolE in DMT1 or TfR1 knockdown cells is unable to reduce apoptosis. The DMT1-KD1 and TfR1-KD1 cells were transfected with pCDNA3-2xFlag or pCDNA3-PolE-2xFlag plasmid, respectively, to obtain PolE overexpression cell lines (DMT1-KD1+PolE and TfR1-KD1+PolE). Then, these two cell lines, together with NP, DMT1-KD1 and TfR1-KD1 cells were subjected to immunoblots to examine protein levels of PolE, PolE2, PolE3, Caspase-3, Caspase-8 and PARP. GAPDH was used as a loading control (A). The same cells used in (A) were also collected and stained with Annexin V-PE/7-AAD, followed by flow cytometry analysis (B-F). (B) NP cells; (C) DMT1-KD1 cells; (D) DMT1-KD1+PolE cells; (E) TfR1-KD1 cells; and (F) TfR1-KD1+PolE cells.

Article Snippet: The antibodies used in this study included anti-PolE (mouse, ThermoFisher Scientific, USA, Catalog #MA5-13616), anti-PolE2 (rabbit, Sigma, USA, Catalog #HPA027555), anti-PolE3 (rabbit, Sigma, USA, Catalog #SAB2101839), anti-caspase-3 (rabbit, Abcam, USA, Catalog #ab90437), anti-caspase-8 (mouse, Cell Signaling Technology, USA, #9746), anti-PARP (Abcam, USA, Catalog #ab15496) and anti-GAPDH (rabbit, Abcam, USA, Catalog #ab128915).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Staining, Flow Cytometry