skbr3  (ATCC)


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    Structured Review

    ATCC skbr3
    Crossing points in cell lines as determined by real-time PCR. HER2/ neu and β-globin were assayed in the same reaction and detected in different fluorescent channels on the LightCycler. A. β-globin Cps (----) were similar in the three cell lines, HER2/ neu Cps (—–) differed depending on the amount of HER2/ neu amplification. (……) No template; (•) MRC-5; (▴) T47D; (▪) <t>SKBR3.</t> B. Standard curves for HER2/ neu (□) and β-globin (∗). Twofold dilutions of standards were used ranging from 10 6 to 2 × 10 3 copies/μL.
    Skbr3, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Comparison of Two Quantitative Polymerase Chain Reaction Methods for Detecting HER2/neu Amplification"

    Article Title: Comparison of Two Quantitative Polymerase Chain Reaction Methods for Detecting HER2/neu Amplification

    Journal: The Journal of molecular diagnostics : JMD

    doi:

    Crossing points in cell lines as determined by real-time PCR. HER2/ neu and β-globin were assayed in the same reaction and detected in different fluorescent channels on the LightCycler. A. β-globin Cps (----) were similar in the three cell lines, HER2/ neu Cps (—–) differed depending on the amount of HER2/ neu amplification. (……) No template; (•) MRC-5; (▴) T47D; (▪) SKBR3. B. Standard curves for HER2/ neu (□) and β-globin (∗). Twofold dilutions of standards were used ranging from 10 6 to 2 × 10 3 copies/μL.
    Figure Legend Snippet: Crossing points in cell lines as determined by real-time PCR. HER2/ neu and β-globin were assayed in the same reaction and detected in different fluorescent channels on the LightCycler. A. β-globin Cps (----) were similar in the three cell lines, HER2/ neu Cps (—–) differed depending on the amount of HER2/ neu amplification. (……) No template; (•) MRC-5; (▴) T47D; (▪) SKBR3. B. Standard curves for HER2/ neu (□) and β-globin (∗). Twofold dilutions of standards were used ranging from 10 6 to 2 × 10 3 copies/μL.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Derivative melting curves in cell lines as determined by competitive PCR. HER2/ neu and β-globin were assayed with competitors in the same reaction. A: Ratios of HER2/ neu melting-peak areas differed depending on the amount of HER2/ neu amplification. B: Ratios of melting-peak areas were similar in the three cell lines for β-globin. ( - . - ), MRC-5; (- - -), T47D; (—), SKBR3; (….), no template.
    Figure Legend Snippet: Derivative melting curves in cell lines as determined by competitive PCR. HER2/ neu and β-globin were assayed with competitors in the same reaction. A: Ratios of HER2/ neu melting-peak areas differed depending on the amount of HER2/ neu amplification. B: Ratios of melting-peak areas were similar in the three cell lines for β-globin. ( - . - ), MRC-5; (- - -), T47D; (—), SKBR3; (….), no template.

    Techniques Used: Polymerase Chain Reaction, Amplification

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    ATCC reagents sk n sh human
    The α2-activated TLR4 signal controls CRF and TH expression ( A ) Protein extracts from <t>N2a</t> cells mock- or α2-transfected in the presence or absence of pHSVsiTLR4 or pHSVsiNC ( n = 5/group) were immunoblotted with antibodies to CRF, TLR4, or β-Actin, and the results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of CRF and TLR4 are significantly higher in the α2- than in the mock-transfected cells, and upregulation is inhibited by siTLR4 but not siNC (* p ≤ 0.05, *** p ≤ 0.001 by t -test). ( B ) Protein extracts from mock- or TLR4-transfected <t>SK-N-SH</t> cells ( n = 5 each) were immunoblotted with antibodies to pCREB, pPKA, TH, TLR4, or GAPDH, and the results are expressed as GAPDH-normalized densitometric units ± SEM. The levels of pCREB, pPKA and TH are significantly higher in the TLR4- than in the mock-transfected cells. The pCREB and TH upregulation is inhibited by treatment with the PKA-specific inhibitor H89 (10 μM) (* p
    Reagents Sk N Sh Human, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The α2-activated TLR4 signal controls CRF and TH expression ( A ) Protein extracts from N2a cells mock- or α2-transfected in the presence or absence of pHSVsiTLR4 or pHSVsiNC ( n = 5/group) were immunoblotted with antibodies to CRF, TLR4, or β-Actin, and the results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of CRF and TLR4 are significantly higher in the α2- than in the mock-transfected cells, and upregulation is inhibited by siTLR4 but not siNC (* p ≤ 0.05, *** p ≤ 0.001 by t -test). ( B ) Protein extracts from mock- or TLR4-transfected SK-N-SH cells ( n = 5 each) were immunoblotted with antibodies to pCREB, pPKA, TH, TLR4, or GAPDH, and the results are expressed as GAPDH-normalized densitometric units ± SEM. The levels of pCREB, pPKA and TH are significantly higher in the TLR4- than in the mock-transfected cells. The pCREB and TH upregulation is inhibited by treatment with the PKA-specific inhibitor H89 (10 μM) (* p

    Journal: Brain Sciences

    Article Title: The GABAA Receptor α2 Subunit Activates a Neuronal TLR4 Signal in the Ventral Tegmental Area that Regulates Alcohol and Nicotine Abuse

    doi: 10.3390/brainsci8040072

    Figure Lengend Snippet: The α2-activated TLR4 signal controls CRF and TH expression ( A ) Protein extracts from N2a cells mock- or α2-transfected in the presence or absence of pHSVsiTLR4 or pHSVsiNC ( n = 5/group) were immunoblotted with antibodies to CRF, TLR4, or β-Actin, and the results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of CRF and TLR4 are significantly higher in the α2- than in the mock-transfected cells, and upregulation is inhibited by siTLR4 but not siNC (* p ≤ 0.05, *** p ≤ 0.001 by t -test). ( B ) Protein extracts from mock- or TLR4-transfected SK-N-SH cells ( n = 5 each) were immunoblotted with antibodies to pCREB, pPKA, TH, TLR4, or GAPDH, and the results are expressed as GAPDH-normalized densitometric units ± SEM. The levels of pCREB, pPKA and TH are significantly higher in the TLR4- than in the mock-transfected cells. The pCREB and TH upregulation is inhibited by treatment with the PKA-specific inhibitor H89 (10 μM) (* p

    Article Snippet: Cells, Plasmids, Transfection, and Reagents SK-N-SH (human) and N2a (mouse) neuroblastoma, mouse monocyte/macrophage RAW264.7, and rat pancreatic RINm5F cells were from American Type Culture Collection (Manassas, VA, USA).

    Techniques: Expressing, Transfection

    Effect of midkine siRNA on cytoprotection. (a) ELISA assay and (b) western blot were used to confirm decreased expression of midkine in doxorubicin resistant SK-N-SH cells (DoxR) treated with midkine. Culture medium from SK-N-SH wild type (WT), DoxR, DoxR cells treated with scramble sequence siRNA (DoxR-scramble), and DoxR cells treated with siRNA to midkine (DoxR-si-midkine) was harvested after growth for 96 hours. (c) SK-N-SH WT cells were grown in co-culture with WT, DoxR, and DoxR-scramble or DoxR-si-midkine cells. Co-cultures were incubated for 48 hours with or without doxorubicin at 10 −7 M and 10 −6 M. SK-N-SH WT cell survival was then quantified through cell counting after staining with trypan blue. Data represents the average of 4 experiments +/− SE. (d) OSA WT cells were grown in co-culture with OSA WT, DoxR, and DoxR-scramble or DoxR-si-midkine cells. Co-cultures were incubated for 48 hours with or without doxorubicin at 10 −7 M and 10 −6 M. OSA WT cell survival was then quantified through cell counting after staining with trypan blue. Data represents the average of 4 experiments +/− SE.

    Journal: ISRN Oncology

    Article Title: Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

    doi: 10.1155/2013/518637

    Figure Lengend Snippet: Effect of midkine siRNA on cytoprotection. (a) ELISA assay and (b) western blot were used to confirm decreased expression of midkine in doxorubicin resistant SK-N-SH cells (DoxR) treated with midkine. Culture medium from SK-N-SH wild type (WT), DoxR, DoxR cells treated with scramble sequence siRNA (DoxR-scramble), and DoxR cells treated with siRNA to midkine (DoxR-si-midkine) was harvested after growth for 96 hours. (c) SK-N-SH WT cells were grown in co-culture with WT, DoxR, and DoxR-scramble or DoxR-si-midkine cells. Co-cultures were incubated for 48 hours with or without doxorubicin at 10 −7 M and 10 −6 M. SK-N-SH WT cell survival was then quantified through cell counting after staining with trypan blue. Data represents the average of 4 experiments +/− SE. (d) OSA WT cells were grown in co-culture with OSA WT, DoxR, and DoxR-scramble or DoxR-si-midkine cells. Co-cultures were incubated for 48 hours with or without doxorubicin at 10 −7 M and 10 −6 M. OSA WT cell survival was then quantified through cell counting after staining with trypan blue. Data represents the average of 4 experiments +/− SE.

    Article Snippet: Cell Culture and Generation of Doxorubicin Resistant Cell Lines Human neuroblastoma SK-N-SH and osteosarcoma SJSA-1 (OSA) cells were purchased from ATCC (Rockville, MA) and grown in DMEM supplemented with 10% FBS at 37°C in 5% CO2 atmosphere.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Sequencing, Co-Culture Assay, Incubation, Cell Counting, Staining

    Effect of midkine siRNA on doxorubicin resistant cellular response to doxorubicin. siRNA was used to knock down midkine expression in doxorubicin resistant SK-N-SH cells (DoxR) to determine if loss of midkine expression results in restoration of drug sensitivity in the DoxR cells. Wild type (WT), DoxR, DoxR cells treated with scramble sequence RNA (DoxR-scramble) and SK-N-SH DoxR cells treated with siRNA to midkine (DoxR-si-midkine) were cultured with or without doxorubicin at 10 −7 M and 10 −6 M for 24 hours. Cell survival was assayed using trypan blue and cell counting. Data represents the average of 4 experiments +/− SE.

    Journal: ISRN Oncology

    Article Title: Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

    doi: 10.1155/2013/518637

    Figure Lengend Snippet: Effect of midkine siRNA on doxorubicin resistant cellular response to doxorubicin. siRNA was used to knock down midkine expression in doxorubicin resistant SK-N-SH cells (DoxR) to determine if loss of midkine expression results in restoration of drug sensitivity in the DoxR cells. Wild type (WT), DoxR, DoxR cells treated with scramble sequence RNA (DoxR-scramble) and SK-N-SH DoxR cells treated with siRNA to midkine (DoxR-si-midkine) were cultured with or without doxorubicin at 10 −7 M and 10 −6 M for 24 hours. Cell survival was assayed using trypan blue and cell counting. Data represents the average of 4 experiments +/− SE.

    Article Snippet: Cell Culture and Generation of Doxorubicin Resistant Cell Lines Human neuroblastoma SK-N-SH and osteosarcoma SJSA-1 (OSA) cells were purchased from ATCC (Rockville, MA) and grown in DMEM supplemented with 10% FBS at 37°C in 5% CO2 atmosphere.

    Techniques: Expressing, Sequencing, Cell Culture, Cell Counting

    Effect of midkine overexpression on wild type cell survival. A midkine overexpressing SK-N-SH cell line (SK-N-SH HMK) was created as previously described [ 6 ]. (a) Midkine expression in the medium of wild type (WT), doxorubicin resistant (DoxR), and HMK cells were analyzed using western blot. SK-N-SH WT, DoxR, and HMK were cultured in 25 cm flasks to 80% confluence. Medium was then exchanged for 2 mL of low fetal bovine serum culture medium and cultured for an additional 48 hours. Cultured medium was then collected and frozen at −80°C prior to being subjected to western blot. The volume of medium loaded on the gel was normalized based on cell count in the flask prior to medium collection. Cell membranes were isolated through a well-established protocol. We have previously demonstrated that midkine transfected cells (HMK) have acquired some doxorubicin resistance [ 6 ]. The MTT cell survival assay was performed to determine if SK-H-SH HMK cells have acquired resistance to other chemotherapeutic drugs including (b) etoposide and (c) cisplatin. (d) SK-N-SH WT, DoxR, and HMK cells were treated with or without doxorubicin at 10 −7 M for 24 hours prior to collection of cell lysates. Proteins were extracted and probed using western blot for Pgp and β -actin.

    Journal: ISRN Oncology

    Article Title: Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

    doi: 10.1155/2013/518637

    Figure Lengend Snippet: Effect of midkine overexpression on wild type cell survival. A midkine overexpressing SK-N-SH cell line (SK-N-SH HMK) was created as previously described [ 6 ]. (a) Midkine expression in the medium of wild type (WT), doxorubicin resistant (DoxR), and HMK cells were analyzed using western blot. SK-N-SH WT, DoxR, and HMK were cultured in 25 cm flasks to 80% confluence. Medium was then exchanged for 2 mL of low fetal bovine serum culture medium and cultured for an additional 48 hours. Cultured medium was then collected and frozen at −80°C prior to being subjected to western blot. The volume of medium loaded on the gel was normalized based on cell count in the flask prior to medium collection. Cell membranes were isolated through a well-established protocol. We have previously demonstrated that midkine transfected cells (HMK) have acquired some doxorubicin resistance [ 6 ]. The MTT cell survival assay was performed to determine if SK-H-SH HMK cells have acquired resistance to other chemotherapeutic drugs including (b) etoposide and (c) cisplatin. (d) SK-N-SH WT, DoxR, and HMK cells were treated with or without doxorubicin at 10 −7 M for 24 hours prior to collection of cell lysates. Proteins were extracted and probed using western blot for Pgp and β -actin.

    Article Snippet: Cell Culture and Generation of Doxorubicin Resistant Cell Lines Human neuroblastoma SK-N-SH and osteosarcoma SJSA-1 (OSA) cells were purchased from ATCC (Rockville, MA) and grown in DMEM supplemented with 10% FBS at 37°C in 5% CO2 atmosphere.

    Techniques: Over Expression, Expressing, Western Blot, Cell Culture, Cell Counting, Isolation, Transfection, MTT Assay, Clonogenic Cell Survival Assay

    Effect of midkine overexpression on cellular response to doxorubicin in co-culture conditions. (a) GFP-transfected wildtype SK-N-SH cells (GFP-WT) were co-cultured with wild type (WT), doxorubicin resistant (DoxR), or human midkine overexpressing SK-N-SH cells (HMK), incubated with (+) or without (−) doxorubicin at 10 −7 M for 48 hours. Photographs were taken using fluorescence microscopy. (b) The viability percentage of fluorescent cells (GFP-WT) after each treatment was determined through Hoechst 33342 staining and cell counting. Data represent an average of three independent determinations +/− SE.

    Journal: ISRN Oncology

    Article Title: Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

    doi: 10.1155/2013/518637

    Figure Lengend Snippet: Effect of midkine overexpression on cellular response to doxorubicin in co-culture conditions. (a) GFP-transfected wildtype SK-N-SH cells (GFP-WT) were co-cultured with wild type (WT), doxorubicin resistant (DoxR), or human midkine overexpressing SK-N-SH cells (HMK), incubated with (+) or without (−) doxorubicin at 10 −7 M for 48 hours. Photographs were taken using fluorescence microscopy. (b) The viability percentage of fluorescent cells (GFP-WT) after each treatment was determined through Hoechst 33342 staining and cell counting. Data represent an average of three independent determinations +/− SE.

    Article Snippet: Cell Culture and Generation of Doxorubicin Resistant Cell Lines Human neuroblastoma SK-N-SH and osteosarcoma SJSA-1 (OSA) cells were purchased from ATCC (Rockville, MA) and grown in DMEM supplemented with 10% FBS at 37°C in 5% CO2 atmosphere.

    Techniques: Over Expression, Co-Culture Assay, Transfection, Cell Culture, Incubation, Fluorescence, Microscopy, Staining, Cell Counting

    Co-culture effect on cellular response to doxorubicin. Wild type human neuroblastoma (SK-N-SH WT) cells co-cultured with SK-N-SH WT cells (WT/WT) were compared to co-cultures of SK-N-SH WT cells with doxorubicin resistant cells (SK-N-SH DoxR) (WT/DoxR). Co-cultures were treated with and without doxorubicin at 10 −7 or 10 −6 M for 48 hours. Surviving cells were quantified using trypan blue staining. The cell survival ratio represents the number of live WT cells in the drug-treated co-culture divided by the number live cells in the untreated (control), * P

    Journal: ISRN Oncology

    Article Title: Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

    doi: 10.1155/2013/518637

    Figure Lengend Snippet: Co-culture effect on cellular response to doxorubicin. Wild type human neuroblastoma (SK-N-SH WT) cells co-cultured with SK-N-SH WT cells (WT/WT) were compared to co-cultures of SK-N-SH WT cells with doxorubicin resistant cells (SK-N-SH DoxR) (WT/DoxR). Co-cultures were treated with and without doxorubicin at 10 −7 or 10 −6 M for 48 hours. Surviving cells were quantified using trypan blue staining. The cell survival ratio represents the number of live WT cells in the drug-treated co-culture divided by the number live cells in the untreated (control), * P

    Article Snippet: Cell Culture and Generation of Doxorubicin Resistant Cell Lines Human neuroblastoma SK-N-SH and osteosarcoma SJSA-1 (OSA) cells were purchased from ATCC (Rockville, MA) and grown in DMEM supplemented with 10% FBS at 37°C in 5% CO2 atmosphere.

    Techniques: Co-Culture Assay, Cell Culture, Staining

    Aβ induced senescence-associated DNA damage in human neuronal cells. (A,B) Representative images of DAPI and γ-H2AX fluorescent staining in Aβ (5 μM)-treated SK-N-SH cells at 24, 48, and 72 h. The quantificative analysis of γ-H2AX foci number in (B) . The pictures were captured by Leica TCS SP8 WLL. Scale bar, 10 μm. (C,D) Cells were incubated with Aβ for indicated time in SK-N-SH cells and western blot analysis of γ-H2AX protein in cell lysates (C) . Actin was used as a loading control. Quantification of γ-H2AX protein level in SK-N-SH cells (D) . (E,F) Representative images of DAPI and γ-H2AX fluorescent staining in Aβ-treated SH-SY5Y cells at 24, 48, and 72 h. The quantificative analysis of γ-H2AX foci number in SK-N-SH cells (F) . The pictures were captured by Leica TCS SP8 WLL. Scale bar, 10 μm. (G,H) Cells were incubated with Aβ for indicated time in SH-SY5Y cells, and western blot analysis of γ-H2AX protein in cell lysates (G) . Actin was used as a loading control. Quantification of γ-H2AX protein level (H) . (I,J) Representative images of 8-OHdG staining in SK-N-SH cells treated by Aβ (5 μM) at indicated time (I) . The quantification of 8-OHdG fluorescent intensity (J) . The pictures were obtained by Leica TCS SP8 WLL. Scale bar, 50 μm. (K,L) Representative images of 8-OHdG staining in SH-SY5Y cells treated by Aβ (5 μM) at indicated time (K) . The quantification of 8-OHdG fluorescent intensity (L) . The pictures were obtained by Leica TCS SP8 WLL. Scale bar, 50 μm. (M,N) Long-range PCR base-assessment of nDNA (12.2 kb) (M) and nDNA (13.5 kb) (N) damage in SK-N-SH cells incubated with Aβ (5 μM) for 72 h. 175 bp as inner control. (O,P) Long-range PCR base assessment of nDNA (12.2 kb) (O) and nDNA (13.5 kb) (P) damage in SH-SY5Y cells incubated with Aβ (5 μM) for 72 h. 175 bp as an inner control. The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alzheimer’s Amyloid-β Accelerates Human Neuronal Cell Senescence Which Could Be Rescued by Sirtuin-1 and Aspirin

    doi: 10.3389/fncel.2022.906270

    Figure Lengend Snippet: Aβ induced senescence-associated DNA damage in human neuronal cells. (A,B) Representative images of DAPI and γ-H2AX fluorescent staining in Aβ (5 μM)-treated SK-N-SH cells at 24, 48, and 72 h. The quantificative analysis of γ-H2AX foci number in (B) . The pictures were captured by Leica TCS SP8 WLL. Scale bar, 10 μm. (C,D) Cells were incubated with Aβ for indicated time in SK-N-SH cells and western blot analysis of γ-H2AX protein in cell lysates (C) . Actin was used as a loading control. Quantification of γ-H2AX protein level in SK-N-SH cells (D) . (E,F) Representative images of DAPI and γ-H2AX fluorescent staining in Aβ-treated SH-SY5Y cells at 24, 48, and 72 h. The quantificative analysis of γ-H2AX foci number in SK-N-SH cells (F) . The pictures were captured by Leica TCS SP8 WLL. Scale bar, 10 μm. (G,H) Cells were incubated with Aβ for indicated time in SH-SY5Y cells, and western blot analysis of γ-H2AX protein in cell lysates (G) . Actin was used as a loading control. Quantification of γ-H2AX protein level (H) . (I,J) Representative images of 8-OHdG staining in SK-N-SH cells treated by Aβ (5 μM) at indicated time (I) . The quantification of 8-OHdG fluorescent intensity (J) . The pictures were obtained by Leica TCS SP8 WLL. Scale bar, 50 μm. (K,L) Representative images of 8-OHdG staining in SH-SY5Y cells treated by Aβ (5 μM) at indicated time (K) . The quantification of 8-OHdG fluorescent intensity (L) . The pictures were obtained by Leica TCS SP8 WLL. Scale bar, 50 μm. (M,N) Long-range PCR base-assessment of nDNA (12.2 kb) (M) and nDNA (13.5 kb) (N) damage in SK-N-SH cells incubated with Aβ (5 μM) for 72 h. 175 bp as inner control. (O,P) Long-range PCR base assessment of nDNA (12.2 kb) (O) and nDNA (13.5 kb) (P) damage in SH-SY5Y cells incubated with Aβ (5 μM) for 72 h. 175 bp as an inner control. The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Article Snippet: SK-N-SH cells and SH-SY5Y cells were purchased from ATCC.

    Techniques: Staining, Incubation, Western Blot, Polymerase Chain Reaction

    Aβ accelerated cell senescence in human neuronal cells. (A) The representative images of SA-β-gal staining in SK-N-SH cells treated by Aβ (5 μM) at indicated time, and quantification of relative number of SA-β-gal-positive cells. The images were captured by Olympus IX73. Scale bars, 50 μm. (B) The representative images of SA-β-gal staining in SH-SY5Y cells challenged by Aβ (5 μM) at indicated time, and quantification of relative number of SA-β-gal-positive cells. The images were captured by Olympus IX73. Scale bars, 50 μm. (C-H) p21 (C) , PAI-1 (D) , MMP3 (E) , p53 (F) , IL6 (G) , Δ133p53 (H) mRNA levels were measured after treatment with Aβ (5 μM) at indicated time. (I-J) Cell growth was detected by luminescent cell viability assay in SK-N-SH cells (I) and SH-SY5Y cells (J) challenged by Aβ (5 μm) at indicated time. (K-N) Cells were incubated with Aβ 1–42 (5 μM) or Aβ 42–1 (5 μM) for 72 h in SK-N-SH cells, and cell lysates were prepared and analyzed using western blotting with p21, PAI-1, p53 antibody. Actin was used as a loading control. Quantification of relative p21 (L) , PAI-1 (M) , p53 (N) protein level in (K) . (O-R) Cells were incubated with Aβ 1–42 (5 μM) or Aβ 42–1 (5 μM) for 72 h in SH-SY5Y cells, and cell lysates were prepared and analyzed using western blotting with p21, PAI-1, and p53 antibody. Actin was used as a loading control. Quantification of relative p21 (P) , PAI-1 (Q) , p53 (R) protein level in (O) . (S-V) Cells were incubated with Aβ (5 μM) for 48 h in NSC-derived neuronal cells, and cell lysates were prepared and analyzed using western blotting with p21, PAI-1, and p53 antibody. Actin was used as a loading control. Quantification of relative p21 (T) , PAI-1 (U) , p53 (V) protein level in (S) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alzheimer’s Amyloid-β Accelerates Human Neuronal Cell Senescence Which Could Be Rescued by Sirtuin-1 and Aspirin

    doi: 10.3389/fncel.2022.906270

    Figure Lengend Snippet: Aβ accelerated cell senescence in human neuronal cells. (A) The representative images of SA-β-gal staining in SK-N-SH cells treated by Aβ (5 μM) at indicated time, and quantification of relative number of SA-β-gal-positive cells. The images were captured by Olympus IX73. Scale bars, 50 μm. (B) The representative images of SA-β-gal staining in SH-SY5Y cells challenged by Aβ (5 μM) at indicated time, and quantification of relative number of SA-β-gal-positive cells. The images were captured by Olympus IX73. Scale bars, 50 μm. (C-H) p21 (C) , PAI-1 (D) , MMP3 (E) , p53 (F) , IL6 (G) , Δ133p53 (H) mRNA levels were measured after treatment with Aβ (5 μM) at indicated time. (I-J) Cell growth was detected by luminescent cell viability assay in SK-N-SH cells (I) and SH-SY5Y cells (J) challenged by Aβ (5 μm) at indicated time. (K-N) Cells were incubated with Aβ 1–42 (5 μM) or Aβ 42–1 (5 μM) for 72 h in SK-N-SH cells, and cell lysates were prepared and analyzed using western blotting with p21, PAI-1, p53 antibody. Actin was used as a loading control. Quantification of relative p21 (L) , PAI-1 (M) , p53 (N) protein level in (K) . (O-R) Cells were incubated with Aβ 1–42 (5 μM) or Aβ 42–1 (5 μM) for 72 h in SH-SY5Y cells, and cell lysates were prepared and analyzed using western blotting with p21, PAI-1, and p53 antibody. Actin was used as a loading control. Quantification of relative p21 (P) , PAI-1 (Q) , p53 (R) protein level in (O) . (S-V) Cells were incubated with Aβ (5 μM) for 48 h in NSC-derived neuronal cells, and cell lysates were prepared and analyzed using western blotting with p21, PAI-1, and p53 antibody. Actin was used as a loading control. Quantification of relative p21 (T) , PAI-1 (U) , p53 (V) protein level in (S) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Article Snippet: SK-N-SH cells and SH-SY5Y cells were purchased from ATCC.

    Techniques: Staining, Cell Viability Assay, Incubation, Western Blot, Derivative Assay

    Aβ induced neuronal cell senescence through suppressing SIRT1 expression. (A) Cells were incubated with Aβ for 24, 48, and 72 h in SK-N-SH cells, and cell lysates were prepared and analyzed using western blotting with SIRT1 and SIRT5 antibody. Actin was used as a loading control. (B,C) Quantification SIRT1 (B) and SIRT5 (C) protein level in (A) . (D) Cells were challenged with Aβ for 72 h in SH-SY5Y cells, western blot analysis of SIRT1 and SIRT5 protein. Actin was used as a loading control. (E,F) Quantification SIRT1 (E) and SIRT5 (F) protein level in (D) . (G) Cells were treated with Aβ for 48 h in NSC-derived neuronal cells and western blot analysis of SIRT1 and SIRT5. Actin was used as a loading control. (H,I) Quantification SIRT1 (H) and SIRT5 (I) protein level in (G) . (J) Cells were transfected with negative control (NC), siSIRT1-1, siSIRT1-2 in SK-N-SH and SH-SY5Y cells, and harvested after 72 h. Western blot analysis of SIRT1 protein level. Actin was used as a loading control. (K,L) Quantification of SIRT1 protein level in SK-N-SH cells (K) and in SH-SY5Y cells (L) . (M,N) The representative images of SA-β-gal staining in SK-N-SH cells transfected with siSIRT1 and stained after 72 h in (M) . The quantification analysis of SA-β-gal-positive cells in (N) . (O,P) The representative images of SA-β-gal staining in SH-SY5Y cells transfected with siSIRT1 and treated for 72 h in (O) . The quantification analysis of SA-β-gal-positive cells in (P) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alzheimer’s Amyloid-β Accelerates Human Neuronal Cell Senescence Which Could Be Rescued by Sirtuin-1 and Aspirin

    doi: 10.3389/fncel.2022.906270

    Figure Lengend Snippet: Aβ induced neuronal cell senescence through suppressing SIRT1 expression. (A) Cells were incubated with Aβ for 24, 48, and 72 h in SK-N-SH cells, and cell lysates were prepared and analyzed using western blotting with SIRT1 and SIRT5 antibody. Actin was used as a loading control. (B,C) Quantification SIRT1 (B) and SIRT5 (C) protein level in (A) . (D) Cells were challenged with Aβ for 72 h in SH-SY5Y cells, western blot analysis of SIRT1 and SIRT5 protein. Actin was used as a loading control. (E,F) Quantification SIRT1 (E) and SIRT5 (F) protein level in (D) . (G) Cells were treated with Aβ for 48 h in NSC-derived neuronal cells and western blot analysis of SIRT1 and SIRT5. Actin was used as a loading control. (H,I) Quantification SIRT1 (H) and SIRT5 (I) protein level in (G) . (J) Cells were transfected with negative control (NC), siSIRT1-1, siSIRT1-2 in SK-N-SH and SH-SY5Y cells, and harvested after 72 h. Western blot analysis of SIRT1 protein level. Actin was used as a loading control. (K,L) Quantification of SIRT1 protein level in SK-N-SH cells (K) and in SH-SY5Y cells (L) . (M,N) The representative images of SA-β-gal staining in SK-N-SH cells transfected with siSIRT1 and stained after 72 h in (M) . The quantification analysis of SA-β-gal-positive cells in (N) . (O,P) The representative images of SA-β-gal staining in SH-SY5Y cells transfected with siSIRT1 and treated for 72 h in (O) . The quantification analysis of SA-β-gal-positive cells in (P) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Article Snippet: SK-N-SH cells and SH-SY5Y cells were purchased from ATCC.

    Techniques: Expressing, Incubation, Western Blot, Derivative Assay, Transfection, Negative Control, Staining

    Exogenous expression of SIRT1 rescued Aβ-induced cell senescence. (A) Cells were transfected with Vector and SIRT1 plasmid in SK-N-SH cells, and after 24 h transfection, cells were incubated with Aβ or without for another 72 h. Western blot analysis of SIRT1, p21, PAI-1 protein level. Actin was used a loading control. (B-D) Quantification of SIRT1 (B) , p21 (C) , and PAI-1 (D) protein level in (A). (E,F) The representative images of SA-β-gal staining in SK-N-SH cells treated with Aβ or without for 72 h, after transfected with Vector and SIRT1 plasmid for 24 h (E) . Quantification of relative number of SA-β-gal-positive cells in (F) . The pictures were obtained by Olympus IX73. Scale bar, 50 μm. (G,H) The representative images of SA-β-gal staining in SH-SY5Y cells incubated with Aβ or without for 72 h, after transfected with Vector and SIRT1 for 24 h (G) . Quantification of relative number of SA-β-gal-positive cells in (H) . The pictures were obtained by Olympus IX73. Scale bar, 50 μm. (I-J) Representative images of DAPI and γ-H2AX fluorescent staining in SK-N-SH cells transfected with SIRT1 plasmid for 24 h and then incubated with Aβ or without for another 72 h in (I) . The quantification of relative γ-H2AX foci number in SK-N-SH cells in (J) . (K,L) Western blot analysis of γ-H2AX protein in SK-N-SH cells transfected with SIRT1 plasmid for 24 h and then incubated with Aβ or without for another 72 h (K) . The quantification of γ-H2AX protein level in (L) . (M,N) Representative images of DAPI and γ-H2AX in SH-SY5Y cells transfected with SIRT1 plasmid for 24 h and followed with Aβ or without for another 72 h in (M) . The quantification of relative γ-H2AX foci number in (N) . (O,P) Western blot analysis of γ-H2AX protein in SH-SY5Y cells transfected with SIRT1 plasmid for 24 h and followed with Aβ or without for another 72 h (O) . The quantification of γ-H2AX protein level in (P) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alzheimer’s Amyloid-β Accelerates Human Neuronal Cell Senescence Which Could Be Rescued by Sirtuin-1 and Aspirin

    doi: 10.3389/fncel.2022.906270

    Figure Lengend Snippet: Exogenous expression of SIRT1 rescued Aβ-induced cell senescence. (A) Cells were transfected with Vector and SIRT1 plasmid in SK-N-SH cells, and after 24 h transfection, cells were incubated with Aβ or without for another 72 h. Western blot analysis of SIRT1, p21, PAI-1 protein level. Actin was used a loading control. (B-D) Quantification of SIRT1 (B) , p21 (C) , and PAI-1 (D) protein level in (A). (E,F) The representative images of SA-β-gal staining in SK-N-SH cells treated with Aβ or without for 72 h, after transfected with Vector and SIRT1 plasmid for 24 h (E) . Quantification of relative number of SA-β-gal-positive cells in (F) . The pictures were obtained by Olympus IX73. Scale bar, 50 μm. (G,H) The representative images of SA-β-gal staining in SH-SY5Y cells incubated with Aβ or without for 72 h, after transfected with Vector and SIRT1 for 24 h (G) . Quantification of relative number of SA-β-gal-positive cells in (H) . The pictures were obtained by Olympus IX73. Scale bar, 50 μm. (I-J) Representative images of DAPI and γ-H2AX fluorescent staining in SK-N-SH cells transfected with SIRT1 plasmid for 24 h and then incubated with Aβ or without for another 72 h in (I) . The quantification of relative γ-H2AX foci number in SK-N-SH cells in (J) . (K,L) Western blot analysis of γ-H2AX protein in SK-N-SH cells transfected with SIRT1 plasmid for 24 h and then incubated with Aβ or without for another 72 h (K) . The quantification of γ-H2AX protein level in (L) . (M,N) Representative images of DAPI and γ-H2AX in SH-SY5Y cells transfected with SIRT1 plasmid for 24 h and followed with Aβ or without for another 72 h in (M) . The quantification of relative γ-H2AX foci number in (N) . (O,P) Western blot analysis of γ-H2AX protein in SH-SY5Y cells transfected with SIRT1 plasmid for 24 h and followed with Aβ or without for another 72 h (O) . The quantification of γ-H2AX protein level in (P) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Article Snippet: SK-N-SH cells and SH-SY5Y cells were purchased from ATCC.

    Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Staining

    Aspirin upregulated SIRT1 to alleviate Aβ-induced senescence. (A) Cells were incubated with aspirin at indicated dose for 6 h and then challenged with Aβ (5 μM) for another 72 h. Western blot analysis of SIRT1, p21, and PAI-1 protein level. Actin was used a loading control. (B-D) Quantification of SIRT1 (B) , p21 (C) , PAI-1 (D) protein level in (A) . (E,F) The representative images of SA-β-gal staining in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (E) . The quantification of relative number of SA-β-gal-positive cells in (F) . The pictures were captured by Olympus IX73. Scale bars, 50 μm. (G,H) Representative images of γ-H2AX fluorescent staining in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (G) . The quantification of relative γ-H2AX foci number in (H) . (I,J) Western blot analysis of γ-H2AX protein level in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (I) . The quantification of γ-H2AX protein level in (J) . (K,L) The representative images of SA-β-gal staining in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h with EX527 (1 μM) (SIRT1 inhibitor) or without, followed by Aβ (5 μM) challenge for 72 h (K) . The quantification of relative number of SA-β-gal-positive cells in (L) . The pictures were captured by Olympus IX73. Scale bars, 50 μm. (M,N) Western blot analysis of γ-H2AX protein level in SH-SY5Y cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (M) . The quantification of γ-H2AX protein level in (N) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Alzheimer’s Amyloid-β Accelerates Human Neuronal Cell Senescence Which Could Be Rescued by Sirtuin-1 and Aspirin

    doi: 10.3389/fncel.2022.906270

    Figure Lengend Snippet: Aspirin upregulated SIRT1 to alleviate Aβ-induced senescence. (A) Cells were incubated with aspirin at indicated dose for 6 h and then challenged with Aβ (5 μM) for another 72 h. Western blot analysis of SIRT1, p21, and PAI-1 protein level. Actin was used a loading control. (B-D) Quantification of SIRT1 (B) , p21 (C) , PAI-1 (D) protein level in (A) . (E,F) The representative images of SA-β-gal staining in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (E) . The quantification of relative number of SA-β-gal-positive cells in (F) . The pictures were captured by Olympus IX73. Scale bars, 50 μm. (G,H) Representative images of γ-H2AX fluorescent staining in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (G) . The quantification of relative γ-H2AX foci number in (H) . (I,J) Western blot analysis of γ-H2AX protein level in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (I) . The quantification of γ-H2AX protein level in (J) . (K,L) The representative images of SA-β-gal staining in SK-N-SH cells pre-incubated with aspirin (100 μM) for 6 h with EX527 (1 μM) (SIRT1 inhibitor) or without, followed by Aβ (5 μM) challenge for 72 h (K) . The quantification of relative number of SA-β-gal-positive cells in (L) . The pictures were captured by Olympus IX73. Scale bars, 50 μm. (M,N) Western blot analysis of γ-H2AX protein level in SH-SY5Y cells pre-incubated with aspirin (100 μM) for 6 h followed by Aβ (5 μM) challenge for 72 h (M) . The quantification of γ-H2AX protein level in (N) . The data are presented as mean ± SEM, n ≥ 3 independent experiments, * p

    Article Snippet: SK-N-SH cells and SH-SY5Y cells were purchased from ATCC.

    Techniques: Incubation, Western Blot, Staining

    In vitro attenuation of RG/B5-AR in the absence of E151. (A) In the absence of E151, the titers of RG/B5-AR (AR) was lower than those of RG/B5-wt (wt). The passage 2 (P2) viruses wt and AR were titrated in Vero, RD, 3T3-hSCARB2, and SK-N-SH cells in the presence (+) or absence (−) of 100 μM E151. The TCID 50 was calculated based on viral antigen positivity by immunofluorescent assay (IFA) in SK-N-SH and cytopathic effect (CPE) in the other three cell lines at 3 days postinfection in three independent experiments ( n = 3; two-tailed unpaired Student’s t test; N.D., not detectable). (B to D) Attenuation of AR was stable. Plaque morphologies (B) of different passages (P1, P5, P10, and P20) of wt and AR in Vero cells. PFU (D) and plaque diameter (E) of the twentieth passage wt-P20 and AR-P20 are presented as the means ± SEM of three independent experiments ( n = 6 in panel D and n = 30 in panel E; two-tailed unpaired Student’s t test). (E and F) One-step growth kinetics demonstrated the growth attenuation of AR at a low but not a high MOI. Vero cells were infected by wt or AR at an MOI of 5 or 0.01. Viral RNA (E) and VP1 protein (F) were quantified at different hours postinfection by qRT-PCR and Western blot analysis, respectively. Lines in panel E indicate the means ± SEM ( n = 6; two-tailed unpaired Student’s t test). A red rectangle in panel F shows that the VP1 protein expression of AR was less than that of the wt from 24 to 40 h postinfection when the MOI was 0.01.

    Journal: Journal of Virology

    Article Title: A Novel Attenuated Enterovirus A71 Mutant with VP1-V238A,K244R Exhibits Reduced Efficiency of Cell Entry/Exit and Augmented Binding Affinity to Sulfated Glycans

    doi: 10.1128/JVI.01055-21

    Figure Lengend Snippet: In vitro attenuation of RG/B5-AR in the absence of E151. (A) In the absence of E151, the titers of RG/B5-AR (AR) was lower than those of RG/B5-wt (wt). The passage 2 (P2) viruses wt and AR were titrated in Vero, RD, 3T3-hSCARB2, and SK-N-SH cells in the presence (+) or absence (−) of 100 μM E151. The TCID 50 was calculated based on viral antigen positivity by immunofluorescent assay (IFA) in SK-N-SH and cytopathic effect (CPE) in the other three cell lines at 3 days postinfection in three independent experiments ( n = 3; two-tailed unpaired Student’s t test; N.D., not detectable). (B to D) Attenuation of AR was stable. Plaque morphologies (B) of different passages (P1, P5, P10, and P20) of wt and AR in Vero cells. PFU (D) and plaque diameter (E) of the twentieth passage wt-P20 and AR-P20 are presented as the means ± SEM of three independent experiments ( n = 6 in panel D and n = 30 in panel E; two-tailed unpaired Student’s t test). (E and F) One-step growth kinetics demonstrated the growth attenuation of AR at a low but not a high MOI. Vero cells were infected by wt or AR at an MOI of 5 or 0.01. Viral RNA (E) and VP1 protein (F) were quantified at different hours postinfection by qRT-PCR and Western blot analysis, respectively. Lines in panel E indicate the means ± SEM ( n = 6; two-tailed unpaired Student’s t test). A red rectangle in panel F shows that the VP1 protein expression of AR was less than that of the wt from 24 to 40 h postinfection when the MOI was 0.01.

    Article Snippet: Human rhabdomyosarcoma (RD; ATCC CCL-136), human SK-N-SH (ATCC HTB11), African green monkey kidney (Vero; ATCC CCL-81), and mouse NIH/3T3 (ATCC CRL-1658) expressing FLAG-tagged human SCABR2 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS) (DMEM-10, Biowest) and 1× antibiotic-antimycotic (Life Technologies) in a 37°C humidified incubator with 5% CO2 .

    Techniques: In Vitro, Immunofluorescence, Two Tailed Test, Infection, Quantitative RT-PCR, Western Blot, Expressing