Journal: Nature metabolism

Article Title: Cardiac glycosides are broad-spectrum senolytics

doi: 10.1038/s42255-019-0122-z

Figure Lengend Snippet: a-c , Senolytic activity of Ouabain in the context of therapy-induced senescence (doxorubicin, n = 4; palbociclib, n =3) and replicative senescence ( n = 4). Statistical significance was calculated using unpaired two-tailed Student’s t -tests. d , Representative pictures (left) of immunofluorescence (IF) staining for p16 INK4a in PBECs after treatment with ABT-263, Ouabain or vehicle (DMSO). p16 INK4a is stained green. Scale bar, 50 μm. Quantification of total and p16 INK4a -positive PBECs (right, n =6). Statistical significance was calculated using two-way ANOVA (Dunnett’s multiple comparisons test). Red stars refer to comparison of p16 INK4a -positive PBECs, black stars refer to comparison of total PBEC numbers. e-g , Dose response analysis of senolytic activity of digoxin ( e ), digitoxin ( f ) and bufalin ( g ) in IMR90 ER:RAS cells ( n = 4). h , Ouabain treatment of senescent cells induce caspase-3/7 activity. IMR90 ER:RAS were treated with 4-OHT or vehicle (DMSO) for 6 days to induce senescence. 100 nM ouabain was then added together with NucLight Rapid Red reagent for cell labelling and Caspase-3/7 reagent for apoptosis (IncuCyte). Caspase 3/7 activity was measured at 4h intervals ( n = 3). i , Pan-caspase inhibition (20 μM Q-VD-OPh) rescues senolytic activity of cardiac glycosides (50 nM ouabain, 50 nM bufalin, 100 nM digoxin) and 1 μM ABT-263 on IMR90 ER:RAS cells ( n = 5). Statistical significance was calculated using two-way ANOVA (Tukey’s test). All error bars represent mean ± s.d; n represents independent experiments.

Article Snippet: The following compounds were used in this study: ABT-263 (Selleckchem, S1001), Ouabain octahydrate (Sigma-Aldrich, O3125), Diphenyleneiodonium chloride (Sigma-Aldrich, D2926), JFD00244 (Sigma-Aldrich, J4829), CGP-74514A hydrochloride (Sigma-Aldrich, C3353), Etoposide (Sigma-Aldrich, E1383), Palbociclib HCl (Selleckchem, S1116), Digoxin (Sigma-Aldrich, D6003), Digitoxin (MedChemExpress HY-B1357), Bufalin (Sigma-Aldrich, B0261), Q-VD-OPh hydrate (Sigma-Aldrich, SML0063), KCl (BioVision, 2115-100), Doxycycline hydrate (Sigma-Aldrich, D9891), 4-Hydroxytamoxifen (Sigma-Aldrich, H7904), CHIR-99021 (Selleckchem, S2924), JNK-IN-8 (Selleckchem, S4901), BMS-582949 (Selleckchem, S8124), ABT-737 (Selleckchem, S1002), Alisertib (Selleckchem, S1133), Barasertib (Selleckchem, S1147), Tozasertib (Selleckchem, S1048), Doxorubicin hydrochloride (Cayman chemical, 15007), Rotenone (Sigma-Aldrich, R8875), Rottlerin (Sigma-Aldrich, R5648), Calmidazolium chloride (Tocris, 2561), BIX-01294 (Selleckchem, S8006), Mibefadril (Sigma-Aldrich, M5441), Ouabagenin (Santa Cruz, sc-295983), Strophanthidin (Sigma-Aldrich, S6626), Strophanthin K (Sigma-Aldrich, S355445), Liproxstatin-1 (Selleckchem S7699), Necrostatin-1 (Selleckchem S8037), Belnacasan (VX-765; Selleckchem S2228), Curcumin (Sigma-Aldrich, 08511), Sorafenib (Selleckchem S7397).

Techniques: Activity Assay, Two Tailed Test, Immunofluorescence, Staining, Inhibition

Journal: Nature metabolism

Article Title: Cardiac glycosides are broad-spectrum senolytics

doi: 10.1038/s42255-019-0122-z

Figure Lengend Snippet: a-b, Quantification of cell survival by trypan blue staining of Huh7 cells ( a ) and HLF cells ( b ) after treatment with the indicated drug combinations ( n = 3). Timeline of the experiment is shown in . Statistical significance was calculated using unpaired two-tailed Student's t -test. c, Quantification of cell survival of senescent (alisertib, palbociclib) and control (DMSO) SK-Mel-5 melanoma cells ( n = 4). Statistical significance was calculated using two-way ANOVA (Dunnett’s test). d, Quantification of cell survival of senescent (doxorubicin, palbociclib) and control (DMSO) MCF-7 or MCF-7 breast cancer cells infected with a shRNA against TP53 ( n = 4). Statistical significance was calculated using twoway ANOVA (Dunnett’s test). e-f, mRNA expression levels of TP53 in MCF-7 cells (e) and HCT-116 cells ( n = 3). Statistical significance was calculated using unpaired two-tailed, Student’s t-test. Data represent mean ± s.d; n represents independent experiments; ns, not significant.

Article Snippet: The following compounds were used in this study: ABT-263 (Selleckchem, S1001), Ouabain octahydrate (Sigma-Aldrich, O3125), Diphenyleneiodonium chloride (Sigma-Aldrich, D2926), JFD00244 (Sigma-Aldrich, J4829), CGP-74514A hydrochloride (Sigma-Aldrich, C3353), Etoposide (Sigma-Aldrich, E1383), Palbociclib HCl (Selleckchem, S1116), Digoxin (Sigma-Aldrich, D6003), Digitoxin (MedChemExpress HY-B1357), Bufalin (Sigma-Aldrich, B0261), Q-VD-OPh hydrate (Sigma-Aldrich, SML0063), KCl (BioVision, 2115-100), Doxycycline hydrate (Sigma-Aldrich, D9891), 4-Hydroxytamoxifen (Sigma-Aldrich, H7904), CHIR-99021 (Selleckchem, S2924), JNK-IN-8 (Selleckchem, S4901), BMS-582949 (Selleckchem, S8124), ABT-737 (Selleckchem, S1002), Alisertib (Selleckchem, S1133), Barasertib (Selleckchem, S1147), Tozasertib (Selleckchem, S1048), Doxorubicin hydrochloride (Cayman chemical, 15007), Rotenone (Sigma-Aldrich, R8875), Rottlerin (Sigma-Aldrich, R5648), Calmidazolium chloride (Tocris, 2561), BIX-01294 (Selleckchem, S8006), Mibefadril (Sigma-Aldrich, M5441), Ouabagenin (Santa Cruz, sc-295983), Strophanthidin (Sigma-Aldrich, S6626), Strophanthin K (Sigma-Aldrich, S355445), Liproxstatin-1 (Selleckchem S7699), Necrostatin-1 (Selleckchem S8037), Belnacasan (VX-765; Selleckchem S2228), Curcumin (Sigma-Aldrich, 08511), Sorafenib (Selleckchem S7397).

Techniques: Staining, Two Tailed Test, Infection, shRNA, Expressing

Journal: Nature Communications

Article Title: WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

doi: 10.1038/s41467-025-66514-0

Figure Lengend Snippet: a RPE1 TP53 KO PAC KO cells were treated with AZD1775 (250 nM) for 6 h in the presence or absence of ISRIB (1 μM), pulse labeled with EdU, and processed for quantitative image-based cytometry. Cell cycle stage was defined by DNA content and EdU positivity. Each point represents a cell. Two independent replicates are defined by two different colors. Bars represent means of each replicate. Experimental set-up ( b ) and flow cytometry analysis ( c ) of RPE1 TP53 KO ATF4-mScarlet-NLS reporter cells. Cells were treated overnight with nocodazole. Mitotic cells were isolated and replated in the presence or absence of palbociclib. After 2 h, AZD1775 (1 µM) and/or A92 (1 µM) was added for 20 h. Data represent mean ± SD (n = 3). Flow cytometry gating strategy ( d ) and analysis of γH2AX in RPE1 TP53 KO cells after treatment with AZD1775 (1 µM) and/or A92 (1 µM) in the absence ( e , left) or presence ( e , right) of nocodazole. Data represent mean ± SD (n = 4). f Flow cytometry analysis of MPM2-positivity in RPE1 TP53 KO cells after treatment with nocodazole, doxorubicin, AZD1775 (1 µM) and/or A92 (1 µM). Data represent mean ± SD (n = 3). g Flow cytometry analysis of MPM2-positivity in control RPE1 TP53 KO PAC KO and ATF4 KO #1 or GCN2 KO #1 cells after treatment with nocodazole, doxorubicin and AZD1775 (1 µM). Data represent mean ± SD (parental conditions n = 4, KO conditions n = 3). h Immunoblot of resting PBMCs or PBMCs stimulated with anti-CD3/CD28 beads. PBMCs were treated with DMSO, AZD1775 (500 nM) or thapsigargin (500 nM) for 24 h. Representative blot of n = 3 independent experiments. i RPE1 TP53 KO PAC KO and GCN2 KO cells were treated with AZD1775 and/or cisplatin for 5 days. Cell survival was analyzed by MTT conversion. Data represent mean ± SD (n = 4). Statistical analysis for c , e , f , g was performed using unpaired t-tests (two-sided), with p ≤ 0.05 considered significant. All replicates are biological replicates unless indicated otherwise. Source data are provided as a file.

Article Snippet: The chemicals used in this study were AZD1775 (#1494, Axon Medchem or S1525, Selleck Chemicals), Debio 0123 (S9778, Selleckchem), A92 (#2720, Axon Medchem), RP-6306 (gift from Repare Therapeutics), AZD7762 (#1399, Axon Medchem), VE-822 (#2452, Axon Medchem), ZNL 02-096 (7240, Tocris), thapsigargin (T9033, Merck), doxorubicin (sc-280681, Santa Cruz Biotechnology), nocodazole (M1404, Sigma), palbociclib (1505, Axon Medchem), integrated stress response inhibitor (ISRIB; SML0843, Sigma or S7400, Selleckchem), salubrinal (S2923, Selleckchem), GCN2iB (S8929, Selleckchem), neratinib (#1526, Axon Medchem) and Emetine (E521535, Toronto Research Chemicals).

Techniques: Labeling, Cytometry, Flow Cytometry, Isolation, Control, Western Blot

Journal: Scientific Reports

Article Title: Novel method for screening functional antibody with comprehensive analysis of its immunoliposome

doi: 10.1038/s41598-021-84043-w

Figure Lengend Snippet: Enhanced cytotoxicity by doxorubicin-encapsulated 214D8-conjugated liposome. (a) Schematic diagram of cytotoxicity analysis. A172 cells were treated with 3 to 100 µM immunoliposomes encapsulating doxorubicin for 1 h, washed with culture media, incubated at 37 °C for 3 days, and analyzed by WST-1 assay. (b) Schematic diagram of doxorubicin-encapsulated immunoliposomes. Quantitative analysis of the immunoliposomes indicated that approximately 2000 doxorubicin molecules were encapsulated per antibody. (c) Reduced relative cell viability by 214D8-conjugated liposome. Treatment with 100 µM 214D8- or 6E1-conjugated liposomes (n = 5 per administration) resulted in 31% and 29% reduction of relative cell viability. Representative results of duplicate independent experiments. Data represent AVG ± SD.

Article Snippet: The immunoliposomes were mixed with doxorubicin hydrochloride (Toronto Research Chemicals, Inc.; Toronto, Canada) at the concentration of 0.2 mg doxorubicin/1 mg phospholipid , and incubated at 42 °C overnight.

Techniques: Incubation, WST-1 Assay, Liposomes

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Controlled Drug Release and Chemotherapy Response in a Novel Acoustofluidic 3D Tumor Platform.

doi: 10.1002/smll.201503342

Figure Lengend Snippet: Figure 4. Analysis of DNA damage in response to drug exposure in the device. A) Immunostaining of γ -H2AX and DAPI in the microfl uidic device under conditions of FUS- exposure, no-drug release (0.3 × 10 −6 M doxorubicin-TS-liposomes), and FUS-triggered drug release (FUS & drug: 0.3 × 10 −6 M ). Each image corresponds to a single confocal slice. Scale bar corresponds to 100 µm. B) Quantifi cation of staining intensity per cell in (A). Average values ( n = 40), and error bars represent standard deviation. Values normalized to the γ -H2AX intensity for the FUS only condition. Statistically signifi cant differences are highlighted (*** p < 0.001).

Article Snippet: To visualize doxorubicin-induced DNA damage in the glioblastoma cells, the devices were fi xed with 4% paraformaldehyde and stained for DAPI (4′, 6-Diamidino-2-Phenylndole, Invitrogen) and rabbit antihuman H2AX (Cell Signaling #2577, MA, USA) detected with a secondary Alexa594 antirabbit antibody (Invitrogen).

Techniques: Immunostaining, Liposomes, Staining, Standard Deviation

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Controlled Drug Release and Chemotherapy Response in a Novel Acoustofluidic 3D Tumor Platform.

doi: 10.1002/smll.201503342

Figure Lengend Snippet: Figure 5. Localized FUS-triggered drug release and cell response. A) Diffusion simulation of drug distribution at 0 and 60 min after FUS-triggered release. B) Spatial profi le (blue) of simulated drug concentration at t = 60 min after drug release and measured cell death under localized FUS- triggered drug treatment (black bars) show good agreement. Cell death profi le under FUS-treatment only (gray bars) is also shown. Concentration normalized to the maximum value (0.3 × 10 −6 M ). Cell death values are reported as differences from baseline values at the region of no release. Averages across n = 4 devices and error bars are SEM. C) Representative confocal images of doxorubicin uptake (green) in an area of drug release (R) compared to an area of no release (NR), 1 h after FUS-triggered heating. Scale bar corresponds to 50 µm. Quantifi cation of doxorubicin (dose of 3 × 10 −6 M to clearly demonstrate differential effects) uptake in microfl uidic devices. Values are normalized to doxorubicin uptake signal for devices treated with drug only but no FUS treatment. Average values across n = 30 cells are plotted, and error bars represent standard deviation. D) Quantifi cation of DNA-damage response in drug release (R) and no release (NR) locations in devices 4 h after localized FUS treatment and doxorubicin-TS-liposome (0.3 × 10 −6 M ) treatment. Bars represent average across n = 40 cells, and error bars represent standard deviation. Values normalized to the γ -H2AX intensity for the untreated control. Statistically signifi cant differences are highlighted (*** p < 0.001, **** p < 0.0001).

Article Snippet: To visualize doxorubicin-induced DNA damage in the glioblastoma cells, the devices were fi xed with 4% paraformaldehyde and stained for DAPI (4′, 6-Diamidino-2-Phenylndole, Invitrogen) and rabbit antihuman H2AX (Cell Signaling #2577, MA, USA) detected with a secondary Alexa594 antirabbit antibody (Invitrogen).

Techniques: Diffusion-based Assay, Concentration Assay, Standard Deviation, Control

Journal: Journal of the American Chemical Society

Article Title: High-Throughput Aptamer Characterization via Real-Time Nuclease Digestion

doi: 10.1021/jacs.5c17561

Figure Lengend Snippet: Demonstrating the generalizability of RT-Exo to characterize aptamers with distinct structures binding various targets. (A) Structure of the adenosine aptamer conjugate A23T-R91–4bp. (B) RT-Exo assay data showing fluorescence over time at different concentrations of adenosine, and (C) specificity of A23T-R91–4bp as determined using the RT-Exo assay. (D) Structure of the THC aptamer conjugate THC1.2-R91–4bp. (E, F) RT-Exo assay data for THC1.2-R91–4bp (E) binding to THC and (F) specificity. (G) Structure of the cocaine aptamer conjugate MNS4.1-R91–4bp. (H, I) RT-Exo assay data for MNS4.1-R91–4bp (H) binding to cocaine and (I) specificity. THCC: (±)-11-nor-9-carboxy-Δ 9 -THC, CBN: cannabinol, THCV: tetrahydrocannabivarin, THCA: THC carboxylic acid A, CBD: cannabidiol, CBDA: cannabidiolic acid, CBGA: cannabigerolic acid, COC: cocaine, AMP: amphetamine, MTC: methcathinone, PENT: pentylone, CLO: clonazepam, CAF: caffeine, PRO: procaine, ACM: acetaminophen, IBU: ibuprofen, NIC: nicotine, BE: benzoylecgonine, MEPH: mephedrone, MDPV: methylenedioxypyrovalerone, FENT: fentanyl, METH: (+)-methamphetamine, MOR: morphine, MTP: methylphenidate, HER: heroin, PSE: pseudoephedrine, MTD: methadone, BZC: benzocaine, SCP: scopolamine, FLU: fluoxetine, SER: serotonin, DOPA: dopamine, LAC: lactose, MAN: mannitol, LIDO: lidocaine, DPH: diphenhydramine, LEV: levamisole, OXY: oxycodone, QUI: quinine, FUB: AB-FUBINACA, ALP: alprazolam, DIAZ: diazepam.

Article Snippet: Levamisole HCl was purchased from MP Biomedicals.

Techniques: Binding Assay, Fluorescence