Journal: Brain research bulletin

Article Title: P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro.

doi: 10.1016/j.brainresbull.2024.110996

Figure Lengend Snippet: Fig. 1. Expression of P2X7 receptor (P2X7R), SOX2, NANOG and glial fibrillary acidic protein (GFAP) in human glioblastoma cancer stem cells (GSCs). GSC cultures at 80 % confluency were fixed and stained with the following antibodies: goat anti-P2X7R with donkey anti-goat Alexa Fluor 488, rabbit anti-SOX2 with goat anti-rabbit Texas Red-X, rabbit anti-NANOG with goat anti-rabbit Texas Red-X, or rabbit anti-GFAP with goat anti-rabbit Texas Red-X. Cells were also counterstained with DAPI. Images were acquired at 60× magnification with a Nikon A1r fluorescent confocal microscope. Scale bars: P2X7R (left) 50μm, P2X7R (right) 250μm, SOX2 250μm, NANOG 250μm, GFAP 350μm.

Article Snippet: A validated human GSC cell line (Smiley et al., 2021) was obtained from Celprogen (cat no. 36104–41) and grown in Glioblastoma Cancer Stem Cell Complete Serum Free Media (Celprogen cat no. M36104–41) in T75 extracellular matrix-coated flasks (Celprogen, cat no. E36104–41-T75).

Techniques: Expressing, Staining, Microscopy

Journal: Brain research bulletin

Article Title: P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro.

doi: 10.1016/j.brainresbull.2024.110996

Figure Lengend Snippet: Fig. 2. The P2X7 receptor (P2X7R) channel is functional in human glioblastoma cancer stem cells (GSCs). GSCs at 80 % confluency were treated with Fluo-4 AM, a synthetic fluorescent indicator, for 30 minutes. To confirm a P2X7R-specific response, some cells were also treated for 15 minutes with 25μM of the specific P2X7R antagonist, AZ10606120 (AZ). Cells were immersed in Dulbecco’s PBS and imaged live with a Nikon A1r fluorescence confocal microscope at 40× magnification over an 8-minute time-series experiment. BzATP (200μM), a P2X7R agonist, was added at approximately 40 seconds in. P2X7R channel activity was determined as the relative fold change in fluorescence intensity (FI) from the baseline after the addition of bzATP (ΔFI/FIBaseline). A) BzATP stimulation induced P2X7R channel responses. There was a significant decrease in Fluo-4 FI in GSCs treated with AZ. Results were yielded from an unpaired t-test with Welch’s correction; n=12 replicates, t(21.23)=8.569, mean difference=-4.12, 95 % CI=-5.114 to −3.118, ****p<0.0001. Data are presented as mean±SEM. B) Graphical representation of the change in FI across 8 minutes in cells treated with Fluo-4 and cells treated with Fluo-4 + AZ. C) Representative timeseries images of FI across 8 minutes in a sample exposed to Fluo-4. D) Representative timeseries images of FI across 8 minutes in a sample exposed to Fluo-4 and pre-treated with AZ.

Article Snippet: A validated human GSC cell line (Smiley et al., 2021) was obtained from Celprogen (cat no. 36104–41) and grown in Glioblastoma Cancer Stem Cell Complete Serum Free Media (Celprogen cat no. M36104–41) in T75 extracellular matrix-coated flasks (Celprogen, cat no. E36104–41-T75).

Techniques: Functional Assay, Fluorescence, Microscopy, Activity Assay

Journal: Brain research bulletin

Article Title: P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro.

doi: 10.1016/j.brainresbull.2024.110996

Figure Lengend Snippet: Fig. 4. P2X7 receptor (P2X7R) antagonism by AZ10606120 (AZ) and its comparison with conventional temozolomide (TMZ) chemotherapy in human glioblastoma stem cells (GSCs). GSCs were treated at 80 % confluency with 5, 15, 25, 50 or 100μM of AZ, or 50μM of TMZ for 72 hours. The control group were untreated cells. Cells were fixed and stained with DAPI. Images were acquired with a Nikon Ti-E inverted fluorescence motorised microscope at 20× objective. The total number of DAPI+ cells were quantified across 16 random fields for each sample and treatment group. A) There was significant GSC depletion with AZ con centrations of 15μM or greater. Results were yielded from a one-way ANOVA with Dunnett’s T3 post-hoc, F(5, 46.73)=58.35, p<0.0001. N=12 replicates. Untreated versus AZ 5μM: mean difference=1232 cells, 95 % CI=-669.1–3134, p=0.67; Untreated versus AZ 15μM: mean difference=4248 cells 95 % CI=2425–6071; Un treated versus AZ 25μM: mean difference=5150 cells, 95 % CI= 3391–6910; Untreated versus AZ 50μM: mean difference=7065 cells, 95 % CI=5630–8500; Un treated versus AZ 100μM: mean difference=7006 cells, 95 % CI= 5583–8428. ****p<0.0001. B) Comparison of 15μM AZ with TMZ in GSCs. Results were yielded from a one-way ANOVA with Dunnett’s T3 post-hoc, F(3, 95.36)=18.84, p<0.0001. N=32 replicates. Untreated versus AZ 15μM: mean difference=4590 cells, 95 % CI=2215–6964; Untreated versus TMZ: mean difference=2002 cells, 95 % CI=-298.9–4303, p=0.12; Untreated versus AZ+TMZ: mean difference=4645 cells, 95 % CI=2556–6734; AZ 15μM versus TMZ: mean difference=-2587 cells, 95 % CI=-4494 to −680.6; AZ 15μM versus AZ+TMZ: mean difference=55.47 cells, 95 % CI=- 1576–1687, p>0.999; TMZ versus AZ+TMZ: mean difference=2643 cells, 95 % CI=1132–4154 **p=0.003, ****p<0.0001. Data are presented as mean±SEM. C) Image representation of GSC monolayers treated with 15μM AZ, TMZ, AZ+TMZ and control.

Article Snippet: A validated human GSC cell line (Smiley et al., 2021) was obtained from Celprogen (cat no. 36104–41) and grown in Glioblastoma Cancer Stem Cell Complete Serum Free Media (Celprogen cat no. M36104–41) in T75 extracellular matrix-coated flasks (Celprogen, cat no. E36104–41-T75).

Techniques: Comparison, Control, Staining, Fluorescence, Microscopy

Journal: Brain research bulletin

Article Title: P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro.

doi: 10.1016/j.brainresbull.2024.110996

Figure Lengend Snippet: Fig. 6. Annexin V staining in human glioblastoma stem cells (GSCs) following P2X7 receptor (P2X7R) antagonism with AZ10606120 (AZ). GSCs were treated with 15μM of AZ for 72 hours at 80 % confluency. Untreated cells were the controls. Cells were subsequently harvested and stained with Annexin V con jugated to phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD). Data were quantified via flow cytometry. Data are presented as mean±SEM. Annexin V results were yielded from an unpaired t-test with Welch’s correction, N=6 replicates. A) Fold change (FC) in the proportion of viable cells in GSCs treated with AZ relative to untreated cells. The difference was not statistically significant: t(8.05)=0.22, mean difference=-0.01-fold, 95 % CI=-0.17–0.14, p=0.83. B) FC in the proportion of early apoptotic cells in GSCs treated with AZ relative to untreated cells. The difference was not statistically significant: T(8.8)=0.67, mean difference=-0.03-fold, 95 % CI=-0.14–0.08, p=0.52. C) FC in the proportion of necrotic cells in GSCs treated with AZ relative to untreated cells. t(5.3)=5.05, mean difference=0.7-fold, 95 % CI=0.35–1.05, **p=0.003. Data are presented as mean±SEM.

Article Snippet: A validated human GSC cell line (Smiley et al., 2021) was obtained from Celprogen (cat no. 36104–41) and grown in Glioblastoma Cancer Stem Cell Complete Serum Free Media (Celprogen cat no. M36104–41) in T75 extracellular matrix-coated flasks (Celprogen, cat no. E36104–41-T75).

Techniques: Staining, Flow Cytometry

Journal: Brain research bulletin

Article Title: P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro.

doi: 10.1016/j.brainresbull.2024.110996

Figure Lengend Snippet: Fig. 5. P2X7 receptor (P2X7R) inhibition with selective antagonist, AZ10606120 (AZ), induces glioblastoma stem cell (GSC) cytotoxicity. Lactate dehydrogenase (LDH) release following treatment with 15, 25, 50 and 100μM of AZ for 72 hours. Data yielded from a one-way ANOVA with Dunnett’s T3 post-hoc, F(4, 97.33)=52.76, p<0.0001. N=24 replicates. Untreated versus AZ 15μM: mean difference=-1.56, 95 % CI=-1.84 to −1.28; Untreated versus AZ 25μM: mean difference=-0.93, 95 % CI=-1.28 to −0.57; Untreated versus AZ 50μM: mean difference=-1.36, 95 % CI=-1.67 to −1.04; Untreated versus AZ 100μM: mean difference=-1.77, 95 % CI=-2.05 to −1.48. ****p<0.0001. Data are presented as mean±SEM.

Article Snippet: A validated human GSC cell line (Smiley et al., 2021) was obtained from Celprogen (cat no. 36104–41) and grown in Glioblastoma Cancer Stem Cell Complete Serum Free Media (Celprogen cat no. M36104–41) in T75 extracellular matrix-coated flasks (Celprogen, cat no. E36104–41-T75).

Techniques: Inhibition

Journal: Brain research bulletin

Article Title: P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro.

doi: 10.1016/j.brainresbull.2024.110996

Figure Lengend Snippet: Fig. 7. Cleaved caspase-3 staining in human glioblastoma cancer stem cells (GSCs) following P2X7 receptor (P2X7R) inhibition by AZ10606120 (AZ). GSCs at 80 % confluency were fixed and stained with primary rabbit anti-cleaved caspase-3 antibody and secondary goat anti-rabbit Alexa Fluor 488. Cell nuclei were subsequently stained with DAPI. Cells were imaged with a Nikon Ti-E fluorescence microscope at 20× magnification. Mean cleaved caspase-3 fluorescence intensity (FI) was quantified for each treatment group across an average of at least 100 cells. A) There was no significant difference in cleaved caspase-3 FI between GSCs treated with AZ and untreated GSCs: t(5.25)=0.22, mean difference=-12.88, 95 % CI=-164.7–138.9, p=0.84. Results were yielded from an unpaired t-test with Welch’s correction. N=6 replicates. Data are presented as mean±SEM. B) Image representation of cleaved caspase-3 FI in untreated GSCs. C) Image representation of cleaved caspase-3 FI in GSCs treated with AZ 15μM.

Article Snippet: A validated human GSC cell line (Smiley et al., 2021) was obtained from Celprogen (cat no. 36104–41) and grown in Glioblastoma Cancer Stem Cell Complete Serum Free Media (Celprogen cat no. M36104–41) in T75 extracellular matrix-coated flasks (Celprogen, cat no. E36104–41-T75).

Techniques: Staining, Inhibition, Fluorescence, Microscopy

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: A specific dispiropiperazine derivative that arrests cell cycle, induces apoptosis, necrosis and DNA damage

doi: 10.1038/s41598-023-35927-6

Figure Lengend Snippet: IC 50 of SPOPP-3 ( 1 ), SPOPP-5 ( 2 ) and doxorubicin against human cancer cell lines.

Article Snippet: For example, SPOPP-3 has stronger anti-proliferative effect on the faster growing human leukemia cell lines (K562, KG1a, CEM) and glioblastoma cell lines (U251, U87) but has weaker effect on the slower growing HepG2 liver cancer cell line, DU145 prostate cancer cell line, and pancreatic cancer cell lines (MiaPaca2, Panc1) (Table ).

Techniques:

Journal: Journal of Anatomy

Article Title: Expression of SARS‐CoV‐2 entry factors in human oral tissue

doi: 10.1111/joa.13391

Figure Lengend Snippet: RT‐PCR analysis for human tissue and cells. The RT‐PCR analysis for the human tissue of the lingual mucosa and buccal mucosa including normal keratinocytes shows that there are cells expressing ACE2 and TMPRSS2. In human epithelial‐like oral cancer cell lines, the reaction product of RT‐PCR for ACE2 mRNA was detected in HSC3 but not in HSC2 or HSC4. The reaction product for TMPRSS2 mRNA was detected in HSC4 but not in HSC2 or HSC4. In the human glioma cell line LN319, the reaction product for ACE2 mRNA was not detected while the reaction product for TMPRSS2 mRNA was detected. The real time‐PCR analysis shows that a strong expression of ACE2 and TMPRSS2 genes are present in the lingual and buccal mucosa, and the gene expression of both ACE2 and TMPRSS2 was significantly stronger in the lingual mucosa than in buccal mucosa or cell line controls. Target gene cDNA were normalized to β‐actin cDNA. The relative gene expression was expressed as arbitrary units according to the following formula: cDNA amounts of experimental samples/cDNA amounts of samples determined as controls (ACE2: HSC3, TMPRSS2: LN319). Values are means ± SD of 5 data. *Significantly different in ANOVA ( p < 0.01)

Article Snippet: The human glioma cell line LN319 was purchased from Addexbio Technologies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Gene Expression