shrna targeting cpne7  (OriGene)

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 Promotes odontoblastic differentiation of progenitor cells in the dental pulp. (a) Schematic diagram of the live‐imaging in recombinant CPNE7 (rCPNE7)‐treated hDPCs. (b) Live‐cell images of hDPCs cultured in differentiation media for 7 days following 24 h of rCPNE7 treatment. Each frame was collected from a single Z plane, and recorded at 320 min intervals. Odontoblast process‐like structure elongation (white boxed area) is observed in rCPNE7‐treated hDPCs. Scale bars: 50 μm. (c) Snapshots at various points on the Z plane, which were generated in three dimensions using the Imaris software. Localizations of TAU (green sphere), F‐Actin (red), and Microtubule (blue) in hDPCs are shown. Boxed area on the left is shown at a higher magnification on the right. N, Nucleus. (d) Schematic diagram of ex vivo subcutaneous transplantation model. Stem cells were mixed with 100 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) particles alone (control) or with rCPNE7 in a poly ε‐caprolactone (PCL) spaces and transplanted subcutaneously into immunocompromised mice for 6 weeks. (e) H&E staining of the sectioned samples was analyzed histologically. Boxed areas on the left (scale bars: 200 μm) Are shown at higher magnification on the right (scale bars: 200 μm). Black arrowheads indicate newly formed mineralized tissues. The dental pulp stem cells (DPSCs), adipogenic stem cells (ADSCs), bone marrow stem cells (BMSCs), gingival fibroblasts (GFs), and periodontal ligament stem cells (PDLSCs) were mixed with 100 mg of HA/TCP particles alone (control) or with rCPNE7 in a poly ε‐caprolactone (PCL) spaces and transplanted subcutaneously into immunocompromised mice for 6 weeks. (f) Semi‐quantification of the volumes of the newly mineralized tissues. All values represented the mean ± SD of triplicate experiments. ** p < 0.01 and * p < 0.05 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Imaging, Recombinant, Cell Culture, Generated, Software, Ex Vivo, Transplantation Assay, Control, Staining

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 ‐null mice molars demonstrate pathologic dentin‐pulp complex phenotype. (a) Light microscopic images of 6‐month‐old WT and Cpne7 −/− mice molars. Thinner coronal dentin (red arrowheads) through which the underlying pulp chambers are seen in Cpne7 −/− mice. Mx, Maxilla; Mn, Mandible. (b) Micro‐computed tomographic images of WT and Cpne7 −/− mice mandibles manifest thinner coronal dentin (red arrowheads) in Cpne7 −/− mice. Quantitative analysis of coronal dentin thickness in WT and Cpne7 −/− mice molars. (c) 3D reconstruction of pulp spaces and quantitative analysis of pulp space volumes in WT and Cpne7 −/− mice molars. (d) Histological analysis of mandibular first molars of WT and Cpne7 −/− mice at 6 months by H&E staining. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; green arrowhead, entrapped cells; red arrow, cells getting entrapped. (e) Cell counting analysis at the dentin‐pulp interfaces of WT and Cpne7 −/− mice molars. (f) TUNEL staining of dental pulp cells of WT and Cpne7 −/− mice at 6 months. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, pathologic dentin; black arrow, TUNEL‐positive cells. (g) Quantitative analysis of the number of TUNEL‐positive cells in WT and Cpne7 −/− mice molars. (h) Transmission electron microscopic images of dentin‐pulp interfaces in the WT and Cpne7 −/− mice molars. Normal nuclear‐shaped hDPCs (white dotted line) and nuclear‐deformed hDPCs (red dotted line) are observed in WT and Cpne7 −/− mice molars, respectively. D, dentin; Op, odontoblast process; N, nucleus; M, mineralized tissue; Cp, cytoplasmic process. (i) Semi‐quantification of nuclear deformation in WT and Cpne7 −/− odontoblasts. All values represented the mean ± SD of triplicate experiments. ** p < 0.01 and * p < 0.05 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Staining, Cell Counting, TUNEL Assay, Transmission Assay, Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 deletion leads to premature aging of the mouse molar pulp environment. (a) Histological analysis of mandibular first molars of WT and Cpne7 −/− mice at 1 year by H&E staining. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; green arrowhead, entrapped cells; red arrow, cells getting entrapped. (b) Senescence‐associated β‐galactosidase (SA‐ β gal) activity (black arrowhead) is observed in 6‐month‐old WT and Cpne7 −/− mice molar by light microscope. Quantitative analysis of the number of SA‐β gal positive cells in WT and Cpne7 −/− mice molars. Scale bars: 20 μm. Boxed areas are shown at higher magnification. D, Dentin; P, Dental pulp; PD, Pathologic dentin. (c) Human DPCs were transfected with shControl (shCon) or shCpne7 vectors for 48 h and cultured in differentiation medium for up to 21 days. Representative images of SA‐β gal activity in Control, shCon, or shCpne7 groups. (d) The hDPCs (passage 3) were treated with 1 mM H 2 O 2 or H 2 O 2 + rCPNE7 for 30 min or 1 h. The hDPCs (passage 22) were used as positive control. SA‐β gal activity (red arrowhead) is observed in H 2 O 2 only or with rCPNE7‐treated hDPCs. (e) Semi‐quantification of SA‐β gal activity was analyzed. (f) The hDPCs (passage 3) were treated with 1 mM H 2 O 2 or H 2 O 2 + rCPNE7 for 72 h. SA‐β gal activity is observed in H 2 O 2 only or with rCPNE7‐treated hDPCs. (g) Quantitative analysis of the number of SA‐β gal positive cells. (h) Histological analysis of mandibular first molars of WT and Cpne7 TG mice at 23 months by H&E staining. Scale bars: 100 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm. D, Dentin; TD, Tertiary dentin; P, Dental pulp; PD, Pathologic dentin. (i) Quantitative analysis of the pulp space volumes in WT and Cpne7 TG mice molars at 23 months. All values represented the mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Staining, Activity Assay, Light Microscopy, Transfection, Cell Culture, Control, Positive Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 modulates intracellular reactive oxygen species (ROS) level in dental pulp cells. (a) Human DPCs were transfected with shCon or shCpne7; 48 h later, hDPCs were treated with 50 μM or 100 μM H 2 O 2 for 24 h and WST‐8 cell viability assay was performed. Oxidative stress‐induced cytotoxicity was analyzed by WST‐8 cell viability assay in hDPCs. (b) Control, shCon, or shCpne7 group incubated with DCFDA for 1 h and microplate analysis of green fluorescent DCFDA signals. (c) DCFDA signal was analyzed in 6‐month‐old WT and Cpne7 −/− mice molar by confocal microscopy. Scale bars: 50 μm. D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin (d) Semi‐quantification of DCFDA signal was analyzed in 6‐month‐old WT and Cpne7 −/− mice molar. (e) Live cell imaging of base intracellular ROS level in hDPCs through DCFDA staining. (f) Live cell imaging of intracellular ROS level by rCPNE7 treatment 15 min after H 2 O 2 treatment in hDPCs through DCFDA staining. Messenger RNA level of Cpne7 was analyzed by RT‐PCR at 30 min or 1 h after H 2 O 2 100 μM, 500 μM, or 1 mM treatment in hDPCs, respectively. (g) DCFDA fluorescence intensity was analyzed. (h) Light microscopic images of hDPCs treated with H 2 O 2 only or with rCPNE7. (i) Cytotoxicity was analyzed by WST‐8 cell viability assay in hDPCs treated with H 2 O 2 only or with rCPNE7. All values represented the mean ± SD of triplicate experiments. * p < 0.05 and ** p < 0.01 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Transfection, Viability Assay, Control, Incubation, Confocal Microscopy, Live Cell Imaging, Staining, Reverse Transcription Polymerase Chain Reaction, Fluorescence

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 deletion leads to DNA damage accumulation in dental pulp cells. (a–c) Heat map construction for hierarchical clustering and a list of downregulated or upregulated genes in rCPNE7‐treated hDPCs. (d) Human DPCs were transfected with shCon or shCpne7 for 48 h. Timeless and Chd6 mRNA levels were evaluated by qPCR. (e) Representative immunofluorescence images of γ‐H2AX (red) and semi‐quantification in 6‐month‐old WT and Cpne7 −/− mice molar ( n = 3). DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 20 μm. Boxed areas are shown at higher magnification. D, Dentin; Od, Odontoblasts; P, Dental pulp. (f) Semi‐quantification of γ‐H2AX (red) signal was analyzed. (g, h) Representative immunofluorescence images of γ‐H2AX (red) and ROS (green) in hDPCs ( n = 3). DAPI (blue) was counterstained to indicate the nucleus. Semi‐quantification of γ‐H2AX (red) and ROS (green) signal was analyzed in hDPCs. All values represented the mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Transfection, Immunofluorescence, Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: Cpne7 is involved in the repair process of oxidative stress‐induced DNA damage in dental pulp cells. (a) Schematic diagram of the microirradiation model in Cpne7 ‐GFP transfected hDPCs. (b) Expression level of CPNE7 and its localization (white arrowheads) before and after microirradiation in hDPCs. (c) Time course images of CPNE7 recruitment (white arrowheads) to the sites of DNA damage in hDPCs. (d) Semi‐quantification of GFP‐tagged CPNE7 signal at each time point. (e) Schematic diagram of the microirradiation model in hDPCs. (f) The recruitment of endogenous CPNE7 (green) was detected 10 and 30 min after DNA damage induction and co‐localized with γ‐H2AX (red). DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 10 μm. Boxed areas are shown at higher magnification. (g) Representative immunofluorescence images of γ‐H2AX (white arrow) in H 2 O 2 only or with rCPNE7‐treated hDPCs. DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 20 μm. Boxed areas are shown at higher magnification. (h, i) The hDPCs were treated with 5 μM, 100 μM, 1 mM, or 2 mM of H 2 O 2 only or w H 2 O 2 + rCPNE7 for 24 h, respectively. Gamma‐H2AX protein levels were evaluated by western blot analysis and semi‐quantified. (j, k) Neutral comet assay of hDPCs, treated for 24 h with H 2 O 2 only or H 2 O 2 + rCPNE7. Comet images of the hDPCs (green) were observed under a confocal microscope and semi‐quantified. Scale bars: 20 μm. Boxed areas are shown at higher magnification. All values represented the mean ± SD of triplicate experiments. * p < 0.05 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Transfection, Expressing, Immunofluorescence, Western Blot, Neutral Comet Assay, Microscopy, Control

Journal: Aging Cell

Article Title: Cpne7 deficiency induces cellular senescence and premature aging of dental pulp

doi: 10.1111/acel.14061

Figure Lengend Snippet: CPNE7‐derived functional peptide rescues the pathologic dentin‐pulp complex phenotypes in Cpne 7 −/− mice molar. (a) Schematic diagram of the intraperitoneal CPNE7‐derived functional peptide (CPNE7‐DP) injection into WT and Cpne 7 −/− mice. (b) Histological analysis of dental pulp responses with or without intraperitoneal CPNE7‐DP injection in WT and Cpne7 −/− mice 3 months after, by H&E staining. Scale bars: 200 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm. D, dentin; P, pulp; PD, pathologic dentin. (c) Representative immunofluorescence images of γ‐H2AX (red) with or without intraperitoneal CPNE7‐DP injection into 6‐month‐old Cpne7 −/− mice molar. DAPI (blue) was counterstained to indicate the nucleus. Scale bars: 20 μm. Boxed areas were shown at higher magnification. D, Dentin; Od, Odontoblasts; P, Dental pulp. (d) Semi‐quantification of γ‐H2AX (red) signal was analyzed. (e) Schematic diagram of DNA damage rescue model in shCon or shCpne7‐transfected hDPCs. (f, g) CPNE7, γ‐H2AX, and P21 protein levels were evaluated by western blot analysis and semi‐quantified. (h) Histological analysis of dental pulp responses at the cavity preparation sites with or without CPNE7‐DP treatment in WT and Cpne7 −/− mice 3 weeks after H&E staining. *, Defected area; D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; green arrowhead, entrapped cells; red arrow, odontoblast‐like cells. (i) Schematic diagram of the indirect pulp capping model. Defect areas were restored with glass ionomer (GI) cement in WT, Cpne7 −/− , and Cpne7 TG mice at 8 months. (j) Histological analysis of the defected areas after 4 weeks was performed by H&E staining. Scale bars: 500 μm. Boxed areas are shown at higher magnification. Scale bars: 50 μm, 20 μm. *, Defected area; D, Dentin; Od, Odontoblasts; P, Dental pulp; PD, Pathologic dentin; ND, Newly‐formed physiologic dentin. * p < 0.05 and # p < 0.0001 vs. Control.

Article Snippet: Control shRNA (TR20003) and shRNA targeting Cpne7 (TR305242) were purchased from Origene (Rockville, MD).

Techniques: Derivative Assay, Functional Assay, Injection, Staining, Immunofluorescence, Transfection, Western Blot, Control