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: Biofabrication

Article Title: Magnetic molding of tumor spheroids: emerging model for cancer screening.

doi: 10.1088/1758-5090/abc670

Figure Lengend Snippet: Figure 5. Doxorubicin penetration and toxicity in magnetically molded (MM) spheroids. (a) Fluorescence intensity distribution of doxorubicin for spheroids of 1 mm initial size and incubated with 10 µg ml−1 of liposomal doxorubicin for 2 h, 1 d and 3 d. The incubation with the drug was initiated at spheroid maturation times of 4 h, 1 d and 3 d. (b) Doxorubicin distribution in hanging drop sppheroids at day 2 of maturation and incubated for 1 d with the drug. Scale bars = 200 µm. (c) Doxorubicin response curves for cells in 2D culture, for magnetic spheroids of 0.5 mm and 1 mm of initial size and matured for 1 d, and for hanging drop spheroids matured for 2 d. For all conditions, doxorubicin incubation was of 3 d, and cell death % was determined with the alamarBlue™metabolic assay. Data represent mean ± SEM (n = 3).

Article Snippet: Liposomal-encapsulated doxorubicin (300112S-1EA, Avanti® Polar Lipids)was selected to study drug resistance inmagneticallymolded spheroids due to its clinical relevancy.

Techniques: Fluorescence, Incubation, Metabolic Assay