Journal: PLoS ONE

Article Title: The Anti-Tumor Histone Deacetylase Inhibitor SAHA and the Natural Flavonoid Curcumin Exhibit Synergistic Neuroprotection against Amyloid-Beta Toxicity

doi: 10.1371/journal.pone.0085570

Figure Lengend Snippet: A. PC12 cells were treated with different concentrations of Aβ 25–35 (n = 5). *, p <0.05; ***, p <0.001 vs Ctrl. B. PC12 cells were treated with different concentrations of SAHA (n = 5). *, p <0.05; ***, p <0.001 vs Ctrl. C. PC12 cells were treated with different concentrations of curcumin (n = 5). *, p <0.05; ***, p <0.001 vs Ctrl. D. MTT assay was performed to detect cell viability after treating with SAHA and curcumin against Aβ 25–35 -induced cytotoxicity in PC12 cells (n = 5). ***, p <0.001 vs Ctrl; **, p <0.01 vs Aβ 25–35 .

Article Snippet: Rat pheochromocytoma PC12 cells were originally obtained from Chinapeptides Co., Ltd. PC12 neuron cells were cultured in DMEM-F12 containing 7% FBS, 1% penicillin, and 1% streptomycin at 37°C.

Techniques: MTT Assay

Journal: PLoS ONE

Article Title: The Anti-Tumor Histone Deacetylase Inhibitor SAHA and the Natural Flavonoid Curcumin Exhibit Synergistic Neuroprotection against Amyloid-Beta Toxicity

doi: 10.1371/journal.pone.0085570

Figure Lengend Snippet: A. Apoptotic cells were detected by TUNEL assay. Cells were stained with TUNEL positive nuclei (green) and nuclei of PC12 cells (blue). B. The percentage of TUNEL positive cells was determined (n = 5). ***, p <0.001 vs Ctrl; **, p <0.01 vs Aβ 25–35 . Ctrl: the control group. C. Western blot of cleaved caspase 3 (n = 3). ***, p <0.001 vs Ctrl; **, p <0.01 vs Aβ 25–35 . Ctrl: the control group; CASP3: caspase 3.

Article Snippet: Rat pheochromocytoma PC12 cells were originally obtained from Chinapeptides Co., Ltd. PC12 neuron cells were cultured in DMEM-F12 containing 7% FBS, 1% penicillin, and 1% streptomycin at 37°C.

Techniques: TUNEL Assay, Staining, Control, Western Blot

Journal: PLoS ONE

Article Title: The Anti-Tumor Histone Deacetylase Inhibitor SAHA and the Natural Flavonoid Curcumin Exhibit Synergistic Neuroprotection against Amyloid-Beta Toxicity

doi: 10.1371/journal.pone.0085570

Figure Lengend Snippet: A. Representative western blot of the p -Akt (Ser 473) protein expression with SAHA treatment (n = 3). **, p <0.01 vs Ctrl. B. Representative western blot of the p -Akt (Ser 473) protein expression with curcumin treatment (n = 3). **, p <0.01 vs Ctrl. C. Representative western blot of the p -Akt (Ser 473) protein expression with co-treatment of SAHA and curcumin (n = 3) to the PC12 cells (n = 3). *, p <0.05 vs Ctrl; **, p <0.01 vs Aβ 25–35 ; Ctrl: the control group.

Article Snippet: Rat pheochromocytoma PC12 cells were originally obtained from Chinapeptides Co., Ltd. PC12 neuron cells were cultured in DMEM-F12 containing 7% FBS, 1% penicillin, and 1% streptomycin at 37°C.

Techniques: Western Blot, Expressing, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of TNFAIP1 alleviates OGD/R‑induced neuronal damage by suppressing Nrf2/GPX4‑mediated ferroptosis

doi: 10.3892/etm.2022.11724

Figure Lengend Snippet: TNFAIP1 silencing alleviates OGD/R-induced PC12 cell injury. (A) mRNA and (B) protein expression levels of TNFAIP1 in OGD/R-induced PC12 cells were assessed using RT-qPCR and western blotting. (C) mRNA and (D) protein expression levels of TNFAIP1 following transfection with sh-TNFAIP1#1 and 2 were assessed using RT-qPCR and western blotting. (E) Cell proliferation was assessed using Cell Counting Kit-8 assay. (F) Cell apoptosis was assessed using (G) flow cytometry. (H) Western blotting was used to assess expression levels of apoptosis-associated protein. Data are presented as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001. TNFAIP1, TNFα-induced protein 1; OGD/R, oxygen glucose deprivation and reperfusion; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin RNA; NC, negative control; PI, propidium iodide.

Article Snippet: The rat adrenal gland cancer PC12 cell line was purchased from BioVector NTCC.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Cell Counting, Flow Cytometry, Real-time Polymerase Chain Reaction, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of TNFAIP1 alleviates OGD/R‑induced neuronal damage by suppressing Nrf2/GPX4‑mediated ferroptosis

doi: 10.3892/etm.2022.11724

Figure Lengend Snippet: TNFAIP1 silencing decreases oxidative stress and the inflammatory response in OGD/R-induced PC12 cells. (A) DCFH-DA staining was used to assess (B) ROS levels. Scale bar, 50 µm. Levels of (C) GSH and (D) MDA were evaluated using commercially available kits. The protein expression levels of (E) TNF-α, (F) IL-6 and (G) IL-1β were assessed using ELISA. Data are presented as the mean ± SD. ** P<0.01 and *** P<0.001. TNFAIP1, TNFα-induced protein 1; OGD/R, oxygen glucose deprivation and reperfusion; ROS, reactive oxygen species; GSH, glutathione; MDA, malondialdehyde; sh, short hairpin RNA; NC, negative control.

Article Snippet: The rat adrenal gland cancer PC12 cell line was purchased from BioVector NTCC.

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of TNFAIP1 alleviates OGD/R‑induced neuronal damage by suppressing Nrf2/GPX4‑mediated ferroptosis

doi: 10.3892/etm.2022.11724

Figure Lengend Snippet: Knockdown of TNFAIP1 inhibits ferroptosis via activation of the Nrf2 signaling pathway in OGD/R-induced PC12 cells. (A) Western blotting was performed to assess protein expression levels of C-Nrf2, N-Nrf2, HO-1 and NQO-1. (B) Iron Assay kit was used to assess Fe 2+ levels. (C) Western blotting was performed to assess protein expression levels of GPX4, FTH1, FPN and TFR1. Data are presented as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001. TNFAIP1, TNFα-induced protein 1; OGD/R, oxygen glucose deprivation and reperfusion; Nrf2, nuclear factor erythroid 2-related factor 2; C, cytoplasmic; N, nuclear; HO-1, heme oxygenase 1; NQO-1, NADPH quinone dehydrogenase 1; GPX4, glutathione peroxidase 4; FTH1, ferritin heavy chain; FPN, ferroportin; TFR1, transferrin receptor 1; sh, short hairpin RNA; NC, negative control.

Article Snippet: The rat adrenal gland cancer PC12 cell line was purchased from BioVector NTCC.

Techniques: Activation Assay, Western Blot, Expressing, Iron Assay, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of TNFAIP1 alleviates OGD/R‑induced neuronal damage by suppressing Nrf2/GPX4‑mediated ferroptosis

doi: 10.3892/etm.2022.11724

Figure Lengend Snippet: Erastin reverses the effect of TNFAIP1 silencing on OGD/R-induced PC12 cell injury. (A) Cell viability was assessed using Cell Counting Kit-8 assay. (B) Cell apoptosis was assessed using (C) flow cytometry. (D) Western blotting was performed to assess expression levels of apoptosis-associated protein. Data are presented as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001. TNFAIP1, TNFα-induced protein 1; OGD/R, oxygen glucose deprivation and reperfusion; sh, short hairpin RNA; NC, negative control; PI, propidium iodide.

Article Snippet: The rat adrenal gland cancer PC12 cell line was purchased from BioVector NTCC.

Techniques: Cell Counting, Flow Cytometry, Western Blot, Expressing, shRNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Downregulation of TNFAIP1 alleviates OGD/R‑induced neuronal damage by suppressing Nrf2/GPX4‑mediated ferroptosis

doi: 10.3892/etm.2022.11724

Figure Lengend Snippet: Erastin reverses the effects of TNFAIP1 silencing on oxidative stress and inflammation in PC12 cells following OGD/R injury. (A) DCFH-DA staining was performed to assess (B) ROS levels. Scale bar, 50 µm. The levels of (C) GSH and (D) MDA were assessed using commercially available kits. The protein expression levels of (E) TNF-α, (F) IL-6 and (G) IL-1β were assessed using ELISA. Data are presented as the mean ± SD. * P<0.05, ** P<0.01 and *** P<0.001. TNFAIP1, TNFα-induced protein 1; OGD/R, oxygen glucose deprivation and reperfusion; sh, short hairpin RNA; NC, negative control; ROS, reactive oxygen species.

Article Snippet: The rat adrenal gland cancer PC12 cell line was purchased from BioVector NTCC.

Techniques: Staining, Expressing, Enzyme-linked Immunosorbent Assay, shRNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: Inflammatory response and oxidative stress attenuated by sulfiredoxin-1 in neuron-like cells depends on nuclear factor erythroid-2-related factor 2

doi: 10.3892/mmr.2020.11545

Figure Lengend Snippet: SRX1 expression levels are downregulated in PC12 cells stimulated with LPS. (A) Cell Counting Kit-8 assay was used to determine the cell viability of PC12 cells following the stimulation with a range of concentrations of LPS. (B) mRNA and (C) protein expression levels of SRX1 in PC12 cells stimulated with a range of concentrations of LPS were determined using reverse transcription-quantitative PCR and western blotting, respectively. Data are expressed as the mean ± SD from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. SRX1, sulfiredoxin-1; LPS, lipopolysaccharide.

Article Snippet: Rat pheochromocytoma PC12 cells (China Center for Type Culture Collection) were cultured in DMEM (Hyclone; Cytiva), supplemented with 10% FBS (Hyclone; Cytiva), and maintained at 37°C under humid conditions with 5% CO 2 and 95% air.

Techniques: Expressing, Cell Counting, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine Reports

Article Title: Inflammatory response and oxidative stress attenuated by sulfiredoxin-1 in neuron-like cells depends on nuclear factor erythroid-2-related factor 2

doi: 10.3892/mmr.2020.11545

Figure Lengend Snippet: Overexpression of SRX1 reduces the inflammatory response in PC12 cells. (A) Transfection efficiency of Ov-SRX1 in PC12 cells was determined using RT-qPCR. (B) mRNA and (C) protein expression levels of SRX1 in PC12 cells transfected with Ov-SRX1 and stimulated with 5 µg/ml LPS were determined using RT-qPCR and western blotting, respectively. (D) Cell Counting Kit-8 assay was used to determine the cell viability of PC12 cells transfected with Ov-SRX1 and stimulated with 5 µg/ml LPS. (E) TNF-α, IL-1β, IL-18 and IL-10 levels in PC12 cells transfected with Ov-SRX1 and stimulated with 5 µg/ml LPS were detected using ELISA kits. Data are expressed as the mean ± SD from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. SRX1, sulfiredoxin-1; LPS, lipopolysaccharide; Ov, overexpression; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; TNF-α, tumor necrosis factor α; IL, interleukin.

Article Snippet: Rat pheochromocytoma PC12 cells (China Center for Type Culture Collection) were cultured in DMEM (Hyclone; Cytiva), supplemented with 10% FBS (Hyclone; Cytiva), and maintained at 37°C under humid conditions with 5% CO 2 and 95% air.

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Cell Counting, Enzyme-linked Immunosorbent Assay, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Inflammatory response and oxidative stress attenuated by sulfiredoxin-1 in neuron-like cells depends on nuclear factor erythroid-2-related factor 2

doi: 10.3892/mmr.2020.11545

Figure Lengend Snippet: Overexpression of SRX1 relieves oxidative stress in PC12 cells. (A) Intracellular ROS in PC12 cells transfected with Ov-SRX1 and stimulated with 5 µg/ml LPS was stained using 2′,7′-dichlorofluorescin. Scale bar, 50 µm. (B) MDA content and (C) SOD activity in PC12 cells transfected with Ov-SRX1 and stimulated with 5 µg/ml LPS were analyzed using commercial detection kits. (D) Protein expression levels of antioxidative proteins PRDX1, PRDX6, TXNRD1 and SOD2 in PC12 cells transfected with Ov-SRX1 and stimulated with 5 µg/ml LPS were analyzed using western blotting. Data are expressed as the mean ± SD from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. SRX1, sulfiredoxin-1; LPS, lipopolysaccharide; Ov, overexpression; NC, negative control; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase; PRDX, peroxiredoxin; TXNRD1, thioredoxin reductase 1.

Article Snippet: Rat pheochromocytoma PC12 cells (China Center for Type Culture Collection) were cultured in DMEM (Hyclone; Cytiva), supplemented with 10% FBS (Hyclone; Cytiva), and maintained at 37°C under humid conditions with 5% CO 2 and 95% air.

Techniques: Over Expression, Transfection, Staining, Activity Assay, Expressing, Western Blot, Negative Control

Journal: Molecular Medicine Reports

Article Title: Inflammatory response and oxidative stress attenuated by sulfiredoxin-1 in neuron-like cells depends on nuclear factor erythroid-2-related factor 2

doi: 10.3892/mmr.2020.11545

Figure Lengend Snippet: NRF2 controls the expression levels of downstream target genes and SRX1. (A) Expression levels of nuclear NRF2 and downstream target genes NQO1 and HO-1 in PC12 cells stimulated with 5 µg/ml LPS were analyzed using western blotting. (B) mRNA expression levels of SRX1, NQO1 and HO-1 in PC12 cells stimulated with 5 µg/ml LPS with or without 25 µM TBHQ or 5 µM ML385 treatment were determined using reverse transcription-quantitative PCR. (C) Western blotting analysis was performed to analyze the expression levels of nuclear NRF2, NQO1, HO-1 and SRX1 in PC12 cells stimulated with 5 µg/ml LPS with or without 25 µM TBHQ or 5 µM ML385 treatment. Data are expressed as the mean ± SD from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001. SRX1, sulfiredoxin-1; LPS, lipopolysaccharide; Ov, overexpression; NC, negative control; NRF2, nuclear factor erythroid-2-related factor 2; HO-1, heme oxygenase 1; NQO1, NAD(P)H dehydrogenase quinone 1; TBHQ, tert-butylhydroquinone.

Article Snippet: Rat pheochromocytoma PC12 cells (China Center for Type Culture Collection) were cultured in DMEM (Hyclone; Cytiva), supplemented with 10% FBS (Hyclone; Cytiva), and maintained at 37°C under humid conditions with 5% CO 2 and 95% air.

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Over Expression, Negative Control

Journal: The Journal of Neuroscience

Article Title: Apoptosis Induced by p75NTR Overexpression Requires Jun Kinase-Dependent Phosphorylation of Bad

doi: 10.1523/JNEUROSCI.23-36-11373.2003

Figure Lengend Snippet: Overexpression of p75NTR reduces survival in a variety of cell types. A, Cortical neurons; B, PC12; C, U343 (wild-type p53); D, U373 (mutant p53) cells were infected with increasing multiplicities of infection (MOI) of LacZ or p75NTR recombinant adenovirus and then analyzed for survival by the MTT assay (see Materials and Methods). Error bars indicate SD. Results were analyzed for statistical significance by ANOVA (Tukey's HSD multiple comparison). Statistically significant differences of p < 0.001 are indicated by an asterisk.

Article Snippet: The rat pheochromocytoma cell line PC12 was maintained as previously described ( Roux et al., 2001 ), and the PC12rtTA cell line (PC12) was purchased from Clontech and maintained in 10% CO 2 at 37°C in DMEM supplemented with 10% FCS, 5% horse serum, 2 m m l -glutamine, 100 μg/ml penicillin-streptomycin, and 100 μg/ml G418.

Techniques: Over Expression, Mutagenesis, Infection, Recombinant, MTT Assay

Journal: The Journal of Neuroscience

Article Title: Apoptosis Induced by p75NTR Overexpression Requires Jun Kinase-Dependent Phosphorylation of Bad

doi: 10.1523/JNEUROSCI.23-36-11373.2003

Figure Lengend Snippet: p75NTR activates caspases and induces accumulation of cytosolic cytochrome c. A, Cortical neurons infected with 10, 50, or 100 MOI of LacZ or p75NTR recombinant adenovirus were lysed and analyzed by immunoblot for levels of LacZ, p75NTR, and full-length caspase 9 protein or, using cleavage-specific antibodies, for levels of cleaved caspases 3 and 6 and cleaved PARP. B, U373 cells were infected with 50 or 100 MOI of either LacZ or p75NTR adenovirus for 48 hr or treated with etoposide 50 μm (+), and then lysed and analyzed for increases in cleaved caspase 9. C, E15 cortical neurons, U373, and PC12 cells were left uninfected (0) or were infected with 100 MOI of LacZ (Lz), or p75NTR (p75) recombinant adenovirus. Thirty hours later, cells were fractionated for cytosolic components as described in Materials and Methods. Cytosolic fractions normalized for protein content were analyzed by immunoblotting with an antibody directed against cytochrome c.

Article Snippet: The rat pheochromocytoma cell line PC12 was maintained as previously described ( Roux et al., 2001 ), and the PC12rtTA cell line (PC12) was purchased from Clontech and maintained in 10% CO 2 at 37°C in DMEM supplemented with 10% FCS, 5% horse serum, 2 m m l -glutamine, 100 μg/ml penicillin-streptomycin, and 100 μg/ml G418.

Techniques: Infection, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: Apoptosis Induced by p75NTR Overexpression Requires Jun Kinase-Dependent Phosphorylation of Bad

doi: 10.1523/JNEUROSCI.23-36-11373.2003

Figure Lengend Snippet: p75NTR activates the JNK pathway. A, U373 cells were infected with 0, 50, 100, or 200 MOI of control AdLacZ or with Adp75NTR. B, PC12 cells were injected with 0 or 50 MOI of AdLacZ or Adp75NTR. C, Cortical neurons were infected with 10, 50, or 150 MOI of AdLacZ or Adp75NTR. Lysates were prepared 30-48 hr after infection and examined by immunoblot for LacZ, p75NTR, phosphorylated JNK (pJNK), total JNK (SC-474 for A, CS-9252 for B), phosphorylated c-Jun (pJun), and total c-Jun as indicated.

Article Snippet: The rat pheochromocytoma cell line PC12 was maintained as previously described ( Roux et al., 2001 ), and the PC12rtTA cell line (PC12) was purchased from Clontech and maintained in 10% CO 2 at 37°C in DMEM supplemented with 10% FCS, 5% horse serum, 2 m m l -glutamine, 100 μg/ml penicillin-streptomycin, and 100 μg/ml G418.

Techniques: Infection, Injection, Western Blot

Journal: The Journal of Neuroscience

Article Title: Apoptosis Induced by p75NTR Overexpression Requires Jun Kinase-Dependent Phosphorylation of Bad

doi: 10.1523/JNEUROSCI.23-36-11373.2003

Figure Lengend Snippet: Activation of the JNK pathway is required for p75NTR-mediated caspase activation. Immunoblots for phospho-Ser63 c-Jun (pJun), c-Jun, Flag-JIP, LacZ, p75NTR, phospho-Thr183/Tyr185-JNK (pJNK), JNK, and cleaved caspase 3 were performed as indicated on lysates from U373 cells treated with TNF 20 ng/ml that were either left uninfected (0) or infected with JBD-JIP adenovirus (JBD) at 10 MOI (A), cortical neurons infected with 50 MOI of LacZ or p75NTR adenovirus together with increasing amounts (0, 0.05, 0.5, 2.5 MOI) of JBD-JIP adenovirus (B), and PC12 cells infected with 50 MOI of LacZ or p75NTR adenovirus supplemented with LacZ or JBD-JIP (JBD) adenovirus (both at 5 MOI) (C).

Article Snippet: The rat pheochromocytoma cell line PC12 was maintained as previously described ( Roux et al., 2001 ), and the PC12rtTA cell line (PC12) was purchased from Clontech and maintained in 10% CO 2 at 37°C in DMEM supplemented with 10% FCS, 5% horse serum, 2 m m l -glutamine, 100 μg/ml penicillin-streptomycin, and 100 μg/ml G418.

Techniques: Activation Assay, Western Blot, Infection

Journal: The Journal of Neuroscience

Article Title: Apoptosis Induced by p75NTR Overexpression Requires Jun Kinase-Dependent Phosphorylation of Bad

doi: 10.1523/JNEUROSCI.23-36-11373.2003

Figure Lengend Snippet: p75NTR-induced caspase 3 cleavage does not correlate with phosphorylation of c-Jun. PC12 cells were infected with 50 MOI of AdLacZ (Lz), Adp75NTR (p75), or AdMLK3 (MLK) recombinant adenovirus, and lysates were prepared at 30 hr after infection (A, C), or at 12, 18, 24, and 30 hr after infection (B). Lysates normalized for protein content were analyzed for LacZ, p75NTR, cleaved caspase 3, phospho-Thr183/Tyr185-JNK (pJNK), total JNK, phospho-Ser63 c-Jun (pJun), and total c-Jun protein levels by immunoblot as indicated.

Article Snippet: The rat pheochromocytoma cell line PC12 was maintained as previously described ( Roux et al., 2001 ), and the PC12rtTA cell line (PC12) was purchased from Clontech and maintained in 10% CO 2 at 37°C in DMEM supplemented with 10% FCS, 5% horse serum, 2 m m l -glutamine, 100 μg/ml penicillin-streptomycin, and 100 μg/ml G418.

Techniques: Infection, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: Apoptosis Induced by p75NTR Overexpression Requires Jun Kinase-Dependent Phosphorylation of Bad

doi: 10.1523/JNEUROSCI.23-36-11373.2003

Figure Lengend Snippet: p75NTR activates JNK-dependent phosphorylation and oligomerization of Bad. A, U373 cells were infected with 0, 50, 100, or 200 MOI of LacZ or p75NTR adenovirus, and lysates were analyzed by immunoblot for LacZ, p75NTR, phospho-Ser128 Bad, and Bad (C-20, shown; N19, data not shown). B, PC12 cells were left uninfected (0) or were infected with LacZ (Lz) or p75NTR (p75) adenovirus aqt 100 MOI, and lysates were analyzed by immunoblot for LacZ, p75NTR, phospho-Ser128 Bad, and Bad (C-20). C, PC12 cells were infected with nothing (0), LacZ (Lz), or p75NTR (p75) adenovirus together with either 5 MOI of LacZ or JBD-JIP (JBD) adenovirus. Lysates were compared for expression of Bad, cleaved caspase 3, LacZ, p75NTR, and Flag-JIP (Flag) by immunoblot as indicated.

Article Snippet: The rat pheochromocytoma cell line PC12 was maintained as previously described ( Roux et al., 2001 ), and the PC12rtTA cell line (PC12) was purchased from Clontech and maintained in 10% CO 2 at 37°C in DMEM supplemented with 10% FCS, 5% horse serum, 2 m m l -glutamine, 100 μg/ml penicillin-streptomycin, and 100 μg/ml G418.

Techniques: Infection, Western Blot, Expressing

Journal: The Journal of Neuroscience

Article Title: Apoptosis Induced by p75NTR Overexpression Requires Jun Kinase-Dependent Phosphorylation of Bad

doi: 10.1523/JNEUROSCI.23-36-11373.2003

Figure Lengend Snippet: Bad is required for p75NTR-induced apoptosis. PC12 cells were transfected with GFP plasmid alone or with GFP plasmid together with plasmids encoding DN-Bad (S128A) or expressing Bad-RNAi. Cells were infected 48 hr later with LacZ or p75NTR adenovirus and, at 24 hr after infection, were fixed and immunostained for cleaved caspase 3 as described in Materials and Methods. Control experiments established that cleaved caspase 3 immunoreactivity correlates with TUNEL staining and is thus a valid surrogate for direct measurement of apoptosis (see supplementary Fig. 1, available at www.jneurosci.org). Transfected cells were scored for caspase 3 cleavage by a blind observer (n = 300 cells/condition). **Indicates a difference of p < 0.001 between GFP/Mock (Bar 1) and GFP/p75NTR (Bar 5), and *indicates a difference of p < 0.001 between GFP/p75NTR (Bar 5) and both DN-Bad/p75NTR (Bar 6) and with Bad RNAi/p75NTR (Bar 7), indicated by ANOVA.

Article Snippet: The rat pheochromocytoma cell line PC12 was maintained as previously described ( Roux et al., 2001 ), and the PC12rtTA cell line (PC12) was purchased from Clontech and maintained in 10% CO 2 at 37°C in DMEM supplemented with 10% FCS, 5% horse serum, 2 m m l -glutamine, 100 μg/ml penicillin-streptomycin, and 100 μg/ml G418.

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, TUNEL Assay, Staining