Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 4 h exposure with S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 4 h exposure without S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Toxicology Reports

Article Title: Genotoxicity risk assessment of a 7-hydroxymitragynine-enriched Kratom preparation: An integrated in silico and in vitro approach

doi: 10.1016/j.toxrep.2026.102206

Figure Lengend Snippet: Micronucleus test of Kratom leaf extract after 24 h exposure without S9 in TK6 cells. Results are the mean ± SD of 3 independent experiments. Statistical testing with one-way ANOVA and Tukey’s post-hoc test (* p < 0.05).

Article Snippet: The human B lymphoblastoid cell line (TK6) (CRL-8015; batch No. 70045146), purchased from ATCC, was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

Techniques:

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: Increased mitochondrial respiration renders F12-cultured cells BSO-sensitive. ( A, B ) Oxygen consumption rate (left) in F12 or F12AA-cultured A549 (A) or H838 (B) cells treated with 0.5 μM oligomycin (Oligo), 1 μM FCCP, and 0.5 μM rotenone (Rot), as indicated. Graphs (right) showing parameter data extracted from the oxygen consumption rate and extracellular acidification rate (n = 14 in A549 cells; n = 10 in F12 and 4 in F12AA-cultured H838 cells). ( C , D ) Oxygen consumption rate (left) in F12-cultured A549 (C) or H838 (D) cells transfected with control siRNA or siRNA targeting ATF4 mRNA and assayed as in (A, B). Graphs (right) show extracted parameters (n = 15 for A549; n = 21 for ATF4 siRNA and n = 13 for control siRNA in H838). ( E ) MitoSox fluorescence images of A549 (left) and H838 (right) cells cultured in F12 or F12AA medium. Cells were treated with 30 μM BSO for 40 h (A549) or 48 h (H838). Graphs show IncuCyte-based fluorescence quantification over time. Scale bar, 100 μm. ( F ) MitoSox fluorescence images of A549 cells cultured in F12 or F12AA medium and treated for 24h with 50 μM BSO, 50 μM BSO + 20 μM mito-TEMPO (MT), or control. MitoSox was added during the last 90 min of treatment; cells were then washed and quantified by live imaging. The graph shows IncuCyte-based fluorescence quantification. Scale bar, 100 μm; n = 8–10. ( G ) Confocal microscopy images of F12-cultured A549 cells treated with 100 μM BSO for 24h showing reduced (pink) and oxidized (green) BODIPY-C11 in combination with mitotracker deep red (red). Corresponding graphs show pixel-wise colocalization (Mander's coefficient) of oxidized BODIPY-C11 and mitotracker deep red in F12-cultured A549 (left) or H838 (right) cells treated with 100 μM BSO for 24 h or controls. n = 6–9 visual fields in A549 cells and 48 visual fields in H838 cells. Scale bar, 10 μm. ( H ) Oxidized BODIPY-C11 fluorescence images of A549 cells cultured in F12 medium and treated for 12 h with 100 μM BSO, 100 nM rotenone, 100 nM oligomycin, or combinations of BSO + rotenone or BSO + oligomycin, and controls. Graph shows IncuCyte-based fluorescence quantification. Scale bar, 50 μm. ( I ) Oxidized BODIPY-C11 fluorescence images of A549 cells cultured in F12 medium and treated for 24 h with 100 μM BSO, BSO + 20 μM mito-TEMPO (MT), or control. Graph shows IncuCyte-based fluorescence quantification. Scale bar, 100 μm. ( J ) Oxidized MitoPerOx fluorescence images of A549 (left) and H838 (right) cells cultured in F12 or F12AA medium. Cells were treated with 30 μM BSO or control for 48 h. Graphs show IncuCyte-based fluorescence quantification over time. Scale bar, 100 μm. ( K) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 100 nM oligomycin, 100 nM rotenone, 20 μM mito-TEMPO + rotenone, or mito-TEMPO + oligomycin, or control for 72 h. ( L ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 100 nM oligomycin, 100 nM rotenone, 5 μM ferrostatin-1 (FER) + rotenone, 5 μM liproxstatin-1 (LIP) + rotenone, ferrostatin-1 + oligomycin, or liproxstatin-1 + oligomycin, or control for 72 h. ( M ) Dose response curves of F12-cultured A549 cells treated with BSO in combination with 0.5 μM FCCP, or control for 72 h. ( N ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 25 or 50 μM mito-TEMPO, or control for 72 h. Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints unless otherwise indicated. Error bars show SEM. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.

Article Snippet: Cas9-expressing A549 cells were obtained from GeneCopoeia (SL-504; GeneCopoeia, Inc., Rockville, MD) and the mouse KP cell line was obtained from V. Sayin and is described [ ].

Techniques: Cell Culture, Transfection, Control, Fluorescence, Imaging, Confocal Microscopy

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: Culture in F12 medium sensitizes lung cancer cells to BSO. ( A ) Crystal violet staining of A549 cells that were cultured in RPMI or F12 medium, after treatment with 100 μM BSO or vehicle (Ctrl) for 72 h. ( B ) BSO dose response curves for A549, H838, H1299, H23, and H460 cells cultured in RPMI or F12 medium for 72 h. ( C ) Crystal violet staining and quantification of mouse KP cells that were cultured in RPMI or F12 medium in the presence of BSO at the indicated concentrations for 72 h. ( D ) Dose response curves for A549 cells cultured in RPMI or F12 medium and treated with erastin, RSL3, or auranofin for 72 h. Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM.

Article Snippet: Cas9-expressing A549 cells were obtained from GeneCopoeia (SL-504; GeneCopoeia, Inc., Rockville, MD) and the mouse KP cell line was obtained from V. Sayin and is described [ ].

Techniques: Staining, Cell Culture

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: F12 medium sensitizes lung cancer cells to iron-dependent lipid peroxidation . ( A, B ) Quantification by flow cytometry of oxidized BODIPY-C11 fluorescence in A549 (A) or H838 (B) cells that were cultured in RPMI or F12 medium and treated with 100 μM BSO or vehicle for 24 h. ( C ) Dose response curves for F12-cultured A549 and H838 cells treated with BSO in combination with 5 μM liproxstatin-1 (LIP-1), 5 μM ferrostatin-1 (FER-1) or 50 μM α-tocopherol (α-toco) for 72 h. ( D ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM deferoxamine (DFO) for A549 cells and 9 μM for H838 cells for 72 h. ( E ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 5 μM necrostatin-1 (NEC-1) or 10 μM ZVAD-FMK (ZVAD) for 72 h. ( F ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 4 μg/mL certolizumab (CER), 15 μM CU-CPT4a (C4a), 3 μM resatorvid (RST), or 50 μM necrostatin-1 (NEC-1) for 72 h (ND, not done). Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM. ∗∗∗∗P < 0.0001.

Article Snippet: Cas9-expressing A549 cells were obtained from GeneCopoeia (SL-504; GeneCopoeia, Inc., Rockville, MD) and the mouse KP cell line was obtained from V. Sayin and is described [ ].

Techniques: Flow Cytometry, Fluorescence, Cell Culture

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: The sensitizing effect of F12 medium is caused by lower amino acid content. ( A ) Concentrations of reduced glutathione in lysates of A549 cells cultured in RPMI or F12 medium and treated with the indicated concentrations of BSO for 24 h. ( B ) BSO dose response curves for A549 cells cultured in F12 or F12 medium supplemented with 65 mg/L cystine (F12 L-Cys) for 72 h. ( C ) BSO dose response curves for A549 and H838 cells cultured in F12 or F12AA medium, the latter with amino acid concentrations matching those of RPMI (see ), for 72 h. (D) BSO dose response curves for A549 cells cultured in RPMI or RPMIAA medium, the latter with amino acid concentrations matching those of F12 (see ), for 72 h. ( E, F ) GC/MS data for intracellular levels of serine, methionine, isoleucine, and leucine (E) or cysteine, glutamate, and glycine (F) in A549 cells at 1, 6, 24, and 48 h after switching from RPMI medium to F12 or F12AA medium. The cells were maintained in RPMI and then passaged into fresh RPMI for 24 h before being switched to F12, F12AA, or fresh RPMI. (G) GC/MS data showing uptake of serine, leucine, and isoleucine in A549 cells that were cultured in F12 or F12AA medium for 48 h. (H) Heatmap showing BSO dose responses of A549 cells cultured in F12 medium supplemented with the indicated amino acids at final concentrations matching the ones in RPMI (see ). (I) Concentrations of reduced glutathione in lysates of A549 and H838 cells cultured in F12 or F12AA medium and treated with the indicated concentrations of BSO for 24 h. Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM. ∗∗P < 0.01, ∗P < 0.05.

Article Snippet: Cas9-expressing A549 cells were obtained from GeneCopoeia (SL-504; GeneCopoeia, Inc., Rockville, MD) and the mouse KP cell line was obtained from V. Sayin and is described [ ].

Techniques: Cell Culture, Gas Chromatography-Mass Spectrometry

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: The integrated stress response pathway is activated in F12-cultured cells . ( A ) Western blotting of S6, p-S6, 4E-BP1, p-4E-BP1 in protein extracts of A549 cells cultured in F12, F12AA, or RPMI medium and treated with 100 μM BSO for 24 h. HSP90 was used as loading control. (B) Western blotting and quantification of p-S6 and p-4E-BP1 in protein extracts of A549 cells cultured in F12 medium and treated with 10 or 50 nM torin1 for 24 h. HSP90 was used as loading control. ( C ) Viability (luminescence) of F12-cultured A549 cells treated with 10 or 50 nM torin1 for 24 h. ( D ) BSO dose response curves for A549 cells cultured in F12 medium and treated with 10 nM torin1 or control for 72 h. The data were normalized against the mean of the untreated samples for each condition. (E) Schematic of the ISR pathway. ( F, G ) Western blotting and quantification of GCN2, p-GCN2, eIF2α, p-eIF2α, ATF4, and CHOP in protein extracts of A549 (F) or H838 (G) cells cultured in F12 or F12AA medium and treated with 100 μM BSO for 24 h. HSP90 was used as loading control. (H) Western blotting and quantification of p-GCN2 and ATF4 in protein extracts of A549 cells at 0, 6, 9, 12, 24, and 48 h after switching from F12AA medium to a pre-conditioned F12 medium. ( I ) Schematic model showing methionine abundance, estimated methionine abundance, ATF4 expression, and estimated ISR activity, as indicated. Data on methionine abundance were retrieved from E and ATF4 expression from H. Thresholds for mild and robust ISR activation are indicated by arrows. n = 3 replicates for all datapoints, error bars show SEM. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.

Article Snippet: Cas9-expressing A549 cells were obtained from GeneCopoeia (SL-504; GeneCopoeia, Inc., Rockville, MD) and the mouse KP cell line was obtained from V. Sayin and is described [ ].

Techniques: Cell Culture, Western Blot, Control, Expressing, Activity Assay, Activation Assay

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: Increased autophagy in F12-cultured cells does not influence BSO sensitivity . ( A, B ) Western blotting (A) and quantification (B) of LC3B–I and II expression in protein extracts of A549 cells cultured in F12 or F12AA medium and treated with 50 μM chloroquine (ChlQ) for the indicated time periods. HSP90 was used as loading control. (C) Western blotting and quantification of TFRC and Ferritin (heavy chain) in protein extracts of A549 cells cultured in F12 or F12AA medium. Tubulin was used as loading control. (D) Western blotting and quantification of LC3B-II in protein extracts of A549 cells cultured in F12 medium and treated with 20 nM torin1 for 24 h. Tubulin was used as loading control. (E) Dose response curves for F12-cultured A549 cells treated with BSO in combination with 20 nM torin1 or control for 72 h. ( F ) Western blotting and quantification of LC3B-II expression in protein extracts of A549 cells cultured in F12 medium and treated with 0, 0.6, or 5 mM 3-MA in the presence of 50 μM chloroquine (ChlQ) for 1 h. HSP90 was used as loading control. (G) Dose response curves for F12-cultured A549 cells treated with BSO in combination with 0, 0.6, 2.5, or 5 mM 3-MA for 72 h. Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM. ∗∗P < 0.01, ∗P < 0.05.

Article Snippet: Cas9-expressing A549 cells were obtained from GeneCopoeia (SL-504; GeneCopoeia, Inc., Rockville, MD) and the mouse KP cell line was obtained from V. Sayin and is described [ ].

Techniques: Cell Culture, Western Blot, Expressing, Control

Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: Activation of the ISR pathway sensitizes lung cancer cells to BSO. ( A, B ) Western blotting of GCN2 in protein extracts of F12-cultured A549 cells (A) and GCN2, p-eIF2α, ATF4 and CHOP in protein extracts of F12-cultured H838 cells (B) that were transfected with Ctrl siRNA or siRNA targeting GCN2 mRNA. HSP90 was used as loading control. ( C ) BSO dose response curves for A549 (left) and H838 (right) cells cultured in F12 medium for 72 h and transfected with Ctrl siRNA or siRNA targeting GCN2 mRNA. ( D ) Western blotting and quantification of GADD34 in protein extracts of A549 cells that were cultured in F12 or F12AA medium and treated with 100 μM BSO for 24 h. Tubulin was used as loading control. ( E ) Western blotting of GADD34 in protein extracts of F12-cultured A549 cells that were transfected with Ctrl siRNA or siRNA targeting GADD34 mRNA. Tubulin was used as loading control. ( F ) Western blotting and quantification of p-eIF2α and CHOP in protein extracts of F12-cultured A549 cells that were transfected with Ctrl siRNA or siRNA targeting GADD34 mRNA. HSP90 was used as loading control. ( G ) BSO dose response curves for A549 cells cultured in F12 medium for 72 h and transfected with Ctrl siRNA or siRNA targeting GADD34 mRNA. ( H ) Western blotting of ATF4 in protein extracts of F12-cultured A549 (top) or H838 (bottom) cells that were transfected with Ctrl siRNA or siRNA targeting ATF4 mRNA. HSP90 was used as loading control. ( I ) mRNA expression of ASNS, CHAC1, CHOP, and SLC7A11 in F12-cultured A549 cells transfected with Ctrl siRNA or siRNA targeting ATF4 mRNA. GAPDH was used as a reference gene for normalization. ( J ) Western blotting and quantification of ATF4 and CHOP in protein extracts of F12-cultured A549 cells that were transfected with Ctrl siRNA or siRNA targeting ATF4 mRNA. HSP90 was used as loading control. ( K ) BSO dose response curves for A549 (left) and H838 (right) cells cultured in F12 medium for 72 h and transfected with Ctrl siRNA or siRNA targeting ATF4 mRNA. ( L ) Quantification by flow cytometry of oxidized BODIPY-C11 fluorescence in A549 (left) or H838 (right) cells that were cultured in F12 medium and treated with 100 μM BSO or vehicle for 24 h and transfected with Ctrl siRNA or siRNA targeting ATF4 mRNA. ( M ) Western blotting of CHOP in protein extracts of F12-cultured A549 (top) or H838 (bottom) cells that were transfected with Ctrl siRNA or siRNA targeting CHOP mRNA. HSP90 was used as loading control. ( N ) BSO dose response curves for A549 (left) and H838 (right) cells cultured in F12 medium for 72 h and transfected with Ctrl siRNA or siRNA targeting CHOP mRNA. ( O ) Western blotting of ATF4 and CHOP in protein extracts of F12-cultured A549 cells that carried a lentivirus overexpressing CHOP cDNA or control. HSP90 was used as loading control. ( P ) BSO dose response curves for A549 cells that carried lentivirus overexpressing CHOP cDNA or control and were cultured in F12 for 72 h. ( Q ) Western blotting of ATF4 and CHOP in protein extracts of RPMI-cultured A549 cells that carried a lentivirus overexpressing ATF4 cDNA or control. HSP90 was used as loading control. (R) BSO dose response curves for A549 cells that carried a lentivirus overexpressing ATF4 cDNA or control and were cultured in RPMI medium for 72 h. Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints, error bars show SEM. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.

Article Snippet: Cas9-expressing A549 cells were obtained from GeneCopoeia (SL-504; GeneCopoeia, Inc., Rockville, MD) and the mouse KP cell line was obtained from V. Sayin and is described [ ].

Techniques: Activation Assay, Western Blot, Cell Culture, Transfection, Control, Expressing, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: STAR Protocols

Article Title: Protocol to generate human stem cell-derived CD70-directed allogeneic CAR-NKT cells for treating renal cell carcinoma

doi: 10.1016/j.xpro.2025.104340

Figure Lengend Snippet: Evaluation of the in vitro antitumor efficacy of Allo CAR70-NKT cells (A) Experimental design to study the in vitro antitumor efficacy of Allo CAR70-NKT cells against human RCC cell lines. (B) Schematics showing the indicated human tumor cell lines. (C) Tumor cell killing data at 24 h (n = 4). (D) ELISA analyses of IFN-γ and TNF-α production by Allo CAR70-NKT cells after 24-hour coculture with 786-O-FG cells (n = 4). (E) Experimental design to Study the tumor cell killing mechanisms of Allo CAR70-NKT cells mediated by NKRs. (F) Tumor cell killing data at 24 h (E:T ratio = 0.5:1; n = 4). Representative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Student’s t test (D) or one-way ANOVA (C and F).

Article Snippet: Human renal cell carcinoma cell line 786-O , ATCC , CRL-1932.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay