Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: Scientific Reports

Article Title: Probable involvement of p11 with interferon alpha induced depression

doi: 10.1038/srep17029

Figure Lengend Snippet: ( A,B ) The p11 protein levels were measured using western blots. The p11 protein levels were normalized against GAPDH. The results demonstrated that IFN-α treatment down-regulated the p11 protein levels in SH-sy5y cells. ( A ) Different doses of hIFN-α-2b (0, 50, 500, 1000, 2000, and 3000 IU/mL) for 24 h produced significant dose-dependent decreases in the p11 protein levels. ( B ) Time-dependent (0, 6, 12, 24, 36, and 48 h) effects in IFN-α (1000 IU/mL) treated cells, with reduced p11 protein levels at 24 h and the lowest levels at 36 h. ( C ) Dose-dependent effect of IFN-α on the p11 mRNA levels in SH-sy5y cells, which were normalized against GAPDH. SH-sy5y cells were treated with hIFN-α-2b (0, 50, 500, 1000, 2000, and 3000 IU/mL) for 16 h, and p11 mRNA levels were measured using real-time PCR. The real-time PCR results demonstrated no effects of hINF-α-2b on the p11 mRNA levels. All results were representative of three separate experiments. The data represented the mean ± S.E. compared with the controls. * P < 0.05 and ** P < 0.01.

Article Snippet: Human neuroblastoma cell line (SH-sy5y cell line, Jiahe Biotech Company, Shanghai, China) was cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin at 37 °C, with 5% CO 2 .

Techniques: Western Blot, Produced, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Probable involvement of p11 with interferon alpha induced depression

doi: 10.1038/srep17029

Figure Lengend Snippet: ( A–C ) IFN-α treatment down-regulated the 5-HTR1b and 5-HTR4 protein levels in a dose-dependent manner. The 5-HTR1b and 5-HTR4 protein levels were determined using western blotting. Cells were treated with different doses of hIFN-α-2b (0, 50, 500, 1000, 2000, and 3000 IU/mL; 0 IU/mL as the control) for 24 h. ( A ) The western blots detected a significant dose-dependent decrease in the 5-HTR1b/4 protein levels after hIFN-α-2b treatment. The 5-HTR1b (B) and 5-HTR4 ( C ) protein levels were normalized against GAPDH. ( D,E ) The real-time PCR results show that IFN-α treatment has no influence on the 5-HTR1b/4 mRNA levels in SH-sy5y cells. Cells were treated with different doses of hIFN-α-2b for 16 h. The values were normalized against GAPDH. ( D ) The 5-HTR1b mRNA levels in hIFN-α-2b treatment groups had no significant difference compared with the controls ( P > 0.05). ( E ) The 5-HTR4 mRNA levels in hIFN-α-2b treatment groups had no significant difference compared with the controls ( P > 0.05). All results were representative of three separate experiments. The data represented the mean ± S.E. compared with the controls. * P < 0.05 and ** P < 0.01.

Article Snippet: Human neuroblastoma cell line (SH-sy5y cell line, Jiahe Biotech Company, Shanghai, China) was cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin at 37 °C, with 5% CO 2 .

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Probable involvement of p11 with interferon alpha induced depression

doi: 10.1038/srep17029

Figure Lengend Snippet: ( A ) Western blots showed the 5-HTR1b and 5-HTR4 protein levels in SH-sy5y cells treated with hIFN-α-2b (1000 IU/mL) at various time points (0, 6, 12, 24, 36, and 48 h). ( B,C ) The 5-HTR1b and 5-HTR4 protein levels were normalized against GAPDH. The results showed that the 5-HTR1b ( B ) and 5-HTR4 ( C ) levels declined at 6 h and reached their lowest levels at 24 h. All results were representative of three separate experiments. The data represented the mean ± S.E. compared with the controls. * P < 0.05 and ** P < 0.01.

Article Snippet: Human neuroblastoma cell line (SH-sy5y cell line, Jiahe Biotech Company, Shanghai, China) was cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin at 37 °C, with 5% CO 2 .

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Probable involvement of p11 with interferon alpha induced depression

doi: 10.1038/srep17029

Figure Lengend Snippet: ( A ) SH-sy5y cells were treated with 1000 IU/mL hIFNα-2b, and with an equal volume of PBS as the control. The proteins in cytomembrane of SH-sy5y cells were extracted at 24 h. The p11, 5-HTR1b and 5-HTR4 protein levels were analyzed using western blotting. ( B ) The levels of p11, 5-HTR1b and 5-HTR4 protein in cytomembrane were normalized against Pan-cadherin. Tublin test was performed to eliminate the contamination from cytoplasm to cytomembrane proteins. All results were representative of three separate experiments. The data represented the mean ± S.E. compared with the controls. ** P < 0.01.

Article Snippet: Human neuroblastoma cell line (SH-sy5y cell line, Jiahe Biotech Company, Shanghai, China) was cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin at 37 °C, with 5% CO 2 .

Techniques: Control, Western Blot

Journal: Scientific Reports

Article Title: Probable involvement of p11 with interferon alpha induced depression

doi: 10.1038/srep17029

Figure Lengend Snippet: After plasmid transfection for 24 h, 1000 U/mL hIFNα-2b or PBS was added to the medium. Total proteins were extracted at 48 h. and the p11, 5-HTR1b and 5-HTR4 protein levels were analyzed using western blotting. Total RNA was extracted at 36 h and reverse transcription was applied to produce cDNA. The 5-HTR1b and 5-HTR4 mRNA levels were analyzed using real-time PCR. ( A ) SH-sy5y cells were transfected with p11-pcDNA3.0 containing full-length p11cDNA, with pcDNA3.0 as the control. ( B–D ) The p11 ( B ), 5-HTR1b ( C ), and 5-HTR4 ( D ) protein levels in p11-pcDNA3.0 transfection groups were normalized against GAPDH. ( E ) After PBS treatment, SH-sy5y cells were transfected with p11-miRNA that interfered with the expression of p11, miRNA-control vector as the control. ( F–H ) The p11 ( F ), 5-HTR1b ( G ), and 5-HTR4 ( H ) protein levels in p11-miRNA transfection groups were normalized against GAPDH. ( I,J ) mRNA levels of 5-HTR1b ( I ) and 5-HTR4 ( J ) in SH-sy5y cells in each transfection group was analyzed using real-time PCR and normalized against GAPDH. All results were representative of three separate experiments. The data represented the mean ± S.E. compared with the controls. * P < 0.05, ** P < 0.01 and NS , no significant difference.

Article Snippet: Human neuroblastoma cell line (SH-sy5y cell line, Jiahe Biotech Company, Shanghai, China) was cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/mL streptomycin at 37 °C, with 5% CO 2 .

Techniques: Plasmid Preparation, Transfection, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Expressing

Journal: Toxins

Article Title: Curcumin―The Paradigm of a Multi-Target Natural Compound with Applications in Cancer Prevention and Treatment

doi: 10.3390/toxins2010128

Figure Lengend Snippet: Evaluation by gene expression profiling of the molecular targets of curcumin in cancer cells.

Article Snippet: Superarray , human SK-N-MC neuroblastoma cell line , Curcumin is a potent radiosensitizer that inhibits growth of human neuroblastoma cells and downregulates radiation-induced pro-survival factors implicating NF-kB transcription factor. , [ ] .

Techniques: Gene Expression, Expressing, Activity Assay, Knock-Out, Ubiquitin Proteomics, Quantitative Proteomics, Control, Derivative Assay