Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: IFNs mediate immunotherapy-associated gene expression in tumors and activate immune cells in healthy PBMCs. A qPCR was used to detect the PD-L1 expression in A549 (5 × 10 5 in a 6-well plate) treated with IFNα and IFNγ (20 ng/mL for each, incubated for 2 h). B The expression of the top 8 up-regulated genes from RNAseq analysis (Table S2), including ICAM1, BATF, IRF1, SOCS1, HAPLN3, TAP1, PSMB9, and MAFF, were validated by qPCR analysis in A549 treated with IFNα and IFNγ (20 ng/mL for each, incubated for 2 h). C The healthy PBMCs (1 × 10 6 in a 6-well plate) treated with IFNs (20 ng/mL for each, incubated for 24 h), co-cultured with A549, IFNα- and IFNγ (3 h)-pretreated A549 at a 20:1 ratio, and IFNα, IFNγ, and A549 concurrently treated for 24 h were analyzed by flow cytometry to (D and E) detect the activation marker CD107a levels in CD4 + T, CD8 + T cells, and CD45 + CD3 − (nonT) PBMCs (n = 3). CD4 + T and CD8 + T were gated by staining with anti-CD45-Pacific blue, anti-CD3-APC/Cy7, anti-CD8-Alexa488, and anti-CD4-PE. CD107a was detected using anti-CD107a-BV605. (F) In addition, the activation markers IFNG, and cytotoxic marker granzyme B (GZMB) were detected by qPCR in the collected PBMCs after individual treatments. n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Incubation, RNA sequencing, Cell Culture, Flow Cytometry, Activation Assay, Marker, Staining, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: Radiotherapy increases IFNs and promotes immunogenic activity but distinct gene expression in the IFNγ- and 8 Gy-treated A549 cells. A A549 cells (5 × 10 5 in a 6-well plate) treated with irradiation (0, 1, 2, 4, 8 Gy) and further incubated for 24 h were collected to detect the p53-downstream gene CDKN1A, MDM2, and tumor marker MKI67 by qPCR. Statistical analysis was achieved by one-way ANOVA. B In addition, IFNA and IFNG and their downstream PD-L1 expression in the irradiated A549 cells and C MDM2 inhibitor Nutlin-3a-treated A549 cells were measured by qPCR. D The healthy PBMCs (1 × 10 6 in a 6-well plate) treated with IFNα and IFNγ (20 ng/mL for each) for 2 h and 24 h were collected and analyzed for the immune activation marker IFNG and cytotoxic marker granzyme B (GZMB) expression using qPCR. E M1 markers TNFA and CXCL10, and M2 markers ARG1 and IL-10 were also investigated in the collected PBMCs with the individual treatments by qPCR. F The healthy PBMCs (1 × 10 6 in a 6-well plate) incubated with irradiation-treated A549 (0, 4, 8 Gy) at a ratio of 20:1 for 24 h were collected and investigated for the immune activation marker IFNG and cytotoxic marker GZMB expression using qPCR. G Meanwhile, M1 markers TNFA, CXCL10, and M2 markers ARG1, IL-10 were also investigated in the collected PBMCs with the individual treatments by qPCR. (H) RNAseq was used to search for the differential genes in the A549 cells (5 × 10 5 in a 6-well plate) treated with IFNγ (20 ng /mL, incubated for 2 h) and x-ray irradiation (8 Gy was selected based on the highest CDKN1A and MDM2 induction, 24 h post-irradiation). There were 75 up-regulated and 12 down-regulated genes selected in IFNγ-treated A549 and 88 up-regulated and 202 down-regulated genes selected in 8 Gy-treated A549 according to log2 fold change > 1 or < -1 with p value < 0.05 (Table S2-4). There was no overlapped gene between IFNγ and 8 Gy treatment. I The 75 and 88 up-regulated genes in IFNγ- and irradiated A549 cells were analyzed by NetworkAnalyst ( https://www.networkanalyst.ca/ ) based on the KEGG dataset, revealing that the differential genes were involved in STATs and p53 signaling pathways, respectively. n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, non-significant

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, Gene Expression, Irradiation, Incubation, Marker, Expressing, Activation Assay, RNA sequencing, Protein-Protein interactions, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: Radiotherapy specifically suppresses TP53-wild type tumors. A Flow cytometry based on fluorescent Annexin V staining was used to detect apoptosis in the irradiation (0, 4, and 8 Gy)-treated TP53-wild type A549, HCT116, and TP53null HCT116 cells (5 × 10 5 in a 6-well plate) post 24 h culture. n = 2. B - C qPCR was used to validate the 13 irradiation-mediated genes from RNAseq (Table S3 and Table S4), including the increased MDM2, CYFIP2, STOM, and the decreased MKI67, CENPE, ARGHGAP11A, BRCA1, ASPM, ALAN, TOP2A, FANCI, TOPBP1, and ECT2, in (B) A549 treated with 8 Gy (24 h post-irradiation), C A549 treated with MDM2 inhibitor Nutlin-3a (10 µg/mL for 24 h). D and E qPCR was also used to detect p53-downstream CDKN1A and the selected 13 genes in TP53-wild type and TP53null HCT116 treated with 8 Gy of irradiation (24 h post-irradiation). n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Staining, Irradiation, RNA sequencing, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: Radiotherapy induces IFNs in TP53-wild type tumors. A qPCR was used to detect the expression of IFNs in TP53-wild type HepG2, HCT116, TP53-mutant PLC5, and TP53null HCT116 (5 × 10 5 in a 6-well plate) treated with 0, 4, 8 Gy of irradiation (24 h post-irradiation). B The IFNγ-mediated up-regulated genes from RNAseq analysis (Table S2), including PD-L1, ICAM1, BATF, IRF1, SOCS1, HAPLN3, TAP1, PSMB9, and MAFF, were investigated by qPCR in TP53-wild type and TP53null HCT116 (5 × 10 5 in a 6-well plate) treated with 8 Gy of irradiation (24 h post-irradiation). C The cultured medium from the irradiated A549 (0, 4, 8 Gy, post 24 h) was collected to treat parental A549 (5 × 10 5 in a 6-well plate) for 2 h. qPCR was used to measure the selected gene expression. n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Mutagenesis, Irradiation, RNA sequencing, Cell Culture, Gene Expression, Two Tailed Test

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: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Exosomal HMGB1 promotes NSCLC progression. (A) Western blot analysis of HMGB1 expression in vector control and HMGB1‐overexpressing (OE) A549 and PC9 cells. (B) Measurement of HMGB1 levels in exosomes derived from vector control and HMGB1 OE A549 and PC9 cells. (C) Cell proliferation assays of vector and HMGB1 OE A549 and PC9 cells. (D) Cell migration assays in vector and HMGB1 OE A549 and PC9 cells. (E) Colony formation assays for vector and HMGB1 OE A549 and PC9 cells. (F) Cell proliferation of A549 and PC9 cells treated with PBS, recombinant HMGB1 (10 or 100 ng) or exosomes derived from vector or HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). (G) Cell migration of A549 and PC9 cells under the same treatment conditions as in (F). (H) Colony formation capacity of A549 and PC9 cells under the same treatment conditions as in (F). (I) Tumour volume in A549‐bearing mice treated with PBS, HMGB1 (10 ng or 100 ng per mouse) or exosomes from vector or HMGB1 OE cells (1 × 10 10 exosomes per mouse). (J) A549 and PC9 cells were seeded at densities of 1 × 10 5 , 1 × 10 6 and 5 × 10 6 cells per six‐well plate and cultured for 3 days. Exosome production was then analysed.

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Derivative Assay, Migration, Recombinant, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Exosomal HMGB1 activates JAK/STAT3 signalling to promote NSCLC progression. (A) Protein–protein interaction (PPI) network analysis of HMGB1 using the STRING database. (B) Western blot analysis of NF‐κB in A549 and PC9 cells treated with PBS, recombinant HMGB1 (100 ng), exosomes from vector cells or exosomes from HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). (C) ELISA quantification of IL‐6 in the supernatant of A549 and PC9 cells under the same treatment conditions as in (B). (D) Immunofluorescence staining of p‐STAT3 of A549 and PC9 cells under the same treatments, including an additional group co‐treated with exosomes from HMGB1 OE cells and NF‐κB inhibitor (50 μM). (E) Cell proliferation of A549 and PC9 cells treated with HMGB1 OE‐derived exosomes alone or in combination with NF‐κB inhibitor (50 μM) or STAT3 inhibitor (20 μM). (F) Cell migration under the same treatment conditions as in (E). (G) Colony formation assays of A549 and PC9 cells under the same treatment conditions as in (E).

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Western Blot, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Derivative Assay, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Targeting HMGB1 signalling improves therapeutic outcomes in NSCLC. (A) Correlation analysis between immune infiltration scores and HMGB1 expression in 491 LUAD and 500 LUSC patients from the TCGA database. (B) Correlation between HMGB1 expression and the distribution of various immune cell subsets in LUAD and LUSC patients. (C, D) THP‐1–derived M0 macrophages were treated with PBS, HMGB1 (10 or 100 ng) or exosomes derived from vector or HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). M1 macrophage markers (CD86, CD80, iNOS) and M2 markers (CD206, IL‐10, Arg1) were quantified by PCR. (E) Lewis tumour‐bearing mice were treated with PBS, HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse, twice per week), anti‐PD‐1 antibody (RMP1‐14, 200 μg per mouse, twice per week) or combination therapy ( n = 5 per group). Tumour volumes and apoptosis levels in tumour tissues (day 25) were assessed. (F) PC9 cells were treated with PBS or exosomes from HMGB1 OE cells (cell‐to‐exosome ratio = 1:10), followed by Osimertinib (50 nM, 48 h), and apoptosis was measured. (G) A549 and PC9 cells were similarly treated with PBS or HMGB1 OE‐derived exosomes, followed by Cisplatin (5 μM, 48 h), and apoptosis was analysed. (H) A549 and PC9 cells were similarly treated with paclitaxel (10 μM, 48 h) under the same conditions, and cell apoptosis was determined. (I) A549‐bearing mice were treated with HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse), followed by PBS, paclitaxel (PTX, 10 mg/kg, twice per week), STAT3 inhibitor (5 mg/kg, twice per week) or combination therapy. (J) Schematic diagram illustrating the proposed mechanism: HMGB1 upregulates TLR4, thereby activating the NF‐κB–IL‐6 axis and stimulating JAK2/STAT3 signalling to promote tumour progression. Concurrently, HMGB1 facilitates M2 macrophage polarisation.

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Derivative Assay, Plasmid Preparation

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Resistance of Lung CSCs to Oxidative Phosphorylation-Mediated ROS Production and Apoptosis Induction. (A) Cell viability in adherent and spheroid A549 cells treated with indicated concentrations of H 2 O 2 for 24 h (B Left) Annexin V-FITC/PI analysis of apoptosis in adherent and spheroid A549 cells following treatment with H 2 O 2 (100 μM or 250 μM) for 24 h (B Right) Quantification of viable cells. (C) Assessment of 8-oxo-dG levels and (D) comet assay analysis for measuring oxidative DNA damage in adherent and spheroid A549 cells treated without or with H 2 O 2 (250 μM) for 1, 3, 6, or 24 h (E Left) Representative time course of oxygen consumption rates (OCR) in adherent and spheroid A549 cells. (E Right) Quantification of different parameters is shown. (F) Fluorescence levels of Mitosox (mitochondrial superoxide indicator), HPF (hydroxyl radical indicator), or DCF (ROS indicator) in adherent and spheroid A549 cells, analyzed by flow cytometry. Data are presented as mean ± SD (student's two-tailed unpaired t -test). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: Human lung cancer cell line A549 was obtained from the ATCC and grown in F–12K (Corning, Manassas, VA, USA).

Techniques: Phospho-proteomics, Single Cell Gel Electrophoresis, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Lung CSCs Resist ROS-Induced Cell Death through ACSL1 and ACSL4-Mediated Lipid Peroxidation. (A) Western blot showing ACSL1, ACSL4, and ACSL3 protein expression in adherent and spheroid cultures. (B) MDA level (left), 4-HNE level (middle), and ratio of C11-BODIPY 581/591 (right) were used to measure lipid peroxidation in spheroid A549 cells treated with H 2 O 2 (250 μM) and vehicle, TC (ACSL inhibitor, 10 μM) or RG (ACSL4 inhibitor, 20 μM) for 24 h (C, D, and E) Cell viability, 8-oxo-dG level, and comet assay in spheroid A549 cells treated with H 2 O 2 (250 μM) and vehicle, TC (10 μM) or RG (20 μM) for 24 h. Data are presented as mean ± SD (one-way ANOVA followed by Tukey's post-hoc test). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: Human lung cancer cell line A549 was obtained from the ATCC and grown in F–12K (Corning, Manassas, VA, USA).

Techniques: Western Blot, Expressing, Single Cell Gel Electrophoresis

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Elevated TAG and Lipid Droplet Levels in Lung CSCs Compared to Ordinary Lung Cancer Cells. (A) Lipidomic analysis reveals the distribution of lipid classes in adherent and spheroid cultures of A549 and H1993 cells, analyzed by MS. (B) Pie charts illustrating the percentage of lipid classes within the total lipids in adherent and spheroid cultures of A549 and H1993 cells. (C) Venn diagram (left) and bar graph (right) showing overlap of lipid species with significant changes across adherent and spheroid cultures as detected by MS. (D) Heat map illustrating the clustering of 98 lipid species with significant changes across the adherent and spheroid cultures of A549 and H1993 cells. (E) Intracellular TAG concentrations analyzed using a TAG determination kit. (F) Ratios of lipid droplets measured using a lipid droplet isolation kit. (G) Cells were costained with BODIPY 493/503 (green) and perilipin 5 (red) in adherent and spheroid A549 cells, with cell nuclei counterstained with DAPI (blue). (H) Representative proximity ligation assay (PLA) images showing interaction between the mitochondria protein mitofusin 2 and the LDs-associated protein perilipin 1. Data are presented as mean ± SD (student's two-tailed unpaired t -test). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: Human lung cancer cell line A549 was obtained from the ATCC and grown in F–12K (Corning, Manassas, VA, USA).

Techniques: Isolation, Proximity Ligation Assay, Two Tailed Test

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Contribution of Oxidized TAG and Lipid Droplet to the Resistance of ROS-Induced Cell Death in Lung CSCs. (A) 1 H NMR spectra obtained from adherent and spheroid A549 cells treated with/without H 2 O 2 (250 μM, 24 h). The proton signal at 5.2–5.3 ppm corresponds to the glycerol backbone of TAG, while the proton signal at 5.6–5.7 ppm represents lipid peroxidation products. The fraction was quantified and normalized by the total protein amount. (B) Detection of the distribution of oxidized lipids (left) and the level of oxidized TAG (right) using LC-MS. (C) Measurement of TAG levels using LC-MS. (D) Levels of lipid droplets (fold change relative to Sph-CTR) after 24-h exposure to the following pro-oxidants: H 2 O 2 (250 μM), tBH (100 μM), ethanol (100 mM), FeCl 2 (0.25 mM) or hypoxia (1 % oxygen). (E) Representative immunofluorescence images showing costaining with C11-BODIPY 581/591 (oxidized, green) and LipidTOX (Lipid droplet, LD, red) in adherent and spheroid A549 cells treated with/without H 2 O 2 (250 μM, 6 h). Cell nuclei were counterstained with DAPI (blue). (F) Representative immunofluorescence images showing costaining with C11-BODIPY 581/591 (oxidized, green) and MitoBright (mitochondrion, Mito, red) in adherent and spheroid A549 cells treated with/without H 2 O 2 (250 μM, 6 h). Cell nuclei were counterstained with DAPI (blue). Arrows indicate colocalization, and pink arrows indicate non-colocalization. (G) Fluorescence levels of Mitosox in adherent and spheroid A549 cells treated with/without H 2 O 2 (250 μM, 6 h), analyzed by flow cytometry. (H) Time-dependent changes in the relative fluorescence intensity (RFI) of JC-1 in adherent and spheroid A549 cells treated with/without H 2 O 2 (250 μM). Sph, Spheroid-A549; Adh, Adherent-A549; Sph-H, Spheroid-A549 + H 2 O 2 . Data are presented as mean ± SD (one-way ANOVA followed by Tukey's post-hoc test is employed in D; two-way ANOVA followed with Tukey's multiple comparison test is employed in G; student's two-tailed unpaired t -test is employed in H). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: Human lung cancer cell line A549 was obtained from the ATCC and grown in F–12K (Corning, Manassas, VA, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Fluorescence, Flow Cytometry, Comparison, Two Tailed Test

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Increased TAG Synthesis in Lung CSCs via Upregulation of DGAT1/2 Expression. (A) Gene ontology (GO) analysis of RNA-sequencing data to identify spheroid-enriched genes between adherent and spheroid culture of A549 and H1993 cells. (B) Genes identified in TAG biosynthesis process term. (C) Western blot showing protein expression related to TAG biosynthesis process in adherent and spheroid culture. (D) Measurement of TAG levels in spheroid A549 cells treated with vehicle, DGAT1i (DGAT1 inhibitor, PF-04620110, 10 μM), DGAT2i (DGAT2 inhibitor, PF-06424439, 10 μM), or DGAT1i + DGAT2i for 24 h. (E) BODIPY 493/503 staining (green) for lipid droplets in spheroid A549 cells treated with DGAT1i + DGAT2i for 24h. Cell nuclei were counterstained with DAPI (blue). (F) Cell viability of spheroid A549 cells incubated without or with H 2 O 2 (250 μM) and/or indicated DGAT inhibitors (10 μM) for 24 h (G, H, and I) TAG, MDA, and 4-HNE levels in spheroid A549 cells incubated without treatment (CTR), treated with H 2 O 2 (250 μM), treated with DGATi (DGAT1 inhibitor at 10 μM + DGAT2 inhibitor at 10 μM), or in combination with H 2 O 2 and DGATi for 24 h. Data are presented as mean ± SD (one-way ANOVA followed by Tukey's post-hoc test). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: Human lung cancer cell line A549 was obtained from the ATCC and grown in F–12K (Corning, Manassas, VA, USA).

Techniques: Expressing, RNA Sequencing, Western Blot, Staining, Incubation

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Inhibition of Tumor Growth and Clinical Implications of DGAT1 and DGAT2 Suppression in Lung Cancer. (A) Cell viability was assessed after irradiation with 2 Gy, 4 Gy, or 6 Gy for 24 h in adherent and spheroid A549 cells. (B) Surviving fractions were calculated in adherent and spheroid A549 cells following irradiation with doses of 2 Gy, 4 Gy, or 6 Gy over a period of 7 days. (C) Cancer cells were subcutaneously injected. When the tumor size reached approximately 50 mm 3 , the mice were subjected to gamma irradiation (3 × 6 Gy) on days 0, 3, and 6, and tumor volumes were measured on the specified days. (D and E) Cell viability and surviving fractions in spheroid A549 cells with DGAT1 and/or DGAT2 inhibitors after irradiation with 6 Gy. (F) Spheroid A549 cells were subcutaneously injected. When the tumor size reached approximately 50 mm 3 , the mice underwent gamma irradiation (3 × 6 Gy) and were orally administered either a vehicle, a DGAT1 inhibitor (10 mg/kg body weight), or a DGAT2 inhibitor (10 mg/kg body weight) on day 0, day 3, and day 6. (G) Sphere formation in spheroid A549 cells with DGAT1 and/or DGAT2 knockdown. (H) The capability of tumor initiation was assessed through the subcutaneous injection of spheroid A549 cells with DGAT1 and/or DGAT2 knockdown. (I) Immunohistochemical staining of DGAT1 and DGAT2 in patients with lung cancer. Case 1 represents a patient with low expression of DGAT1 and DGAT2. Case 2 represents a patient with high expression of DGAT1 and DGAT2. (J) The survival curves of lung cancer patients with or without DGAT1 and DGAT2 expression (n = 59). Significance is calculated using the Kaplan-Meier method and comparisons are performed using the log-rank test. (K) Survival analysis of the 6-gene signature related to TAG synthesis in lung cancer. Data are presented as mean ± SD (student's two-tailed unpaired t -test is employed in A and B; two-way ANOVA followed with Tukey's multiple comparison test is employed in C; one-way ANOVA followed by Tukey's post-hoc test is employed in D, E, F, and G). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: Human lung cancer cell line A549 was obtained from the ATCC and grown in F–12K (Corning, Manassas, VA, USA).

Techniques: Inhibition, Irradiation, Injection, Knockdown, Immunohistochemical staining, Staining, Expressing, Two Tailed Test, Comparison

Journal: Redox Biology

Article Title: YAP/TEAD-activated TAG synthesis and peroxidation in lipid droplets confer ROS resistance in cancer stem cells

doi: 10.1016/j.redox.2025.103968

Figure Lengend Snippet: Regulation of Lipid Metabolism by Cancer Stem Cells via the YAP1/TEAD Pathway. (A) Top pathways identified by KEGG analysis from RNA-sequencing data between adherent and spheroid culture of A549 and H1993 cells. (B) Representative immunofluorescence images showing the subcellular location of pYAP1 (Y357) and TEAD1 in adherent and spheroid A549 cells. Cell nuclei were counterstained with DAPI. (C) Western blot analysis of cytoplasmic (Cyt) and nuclear (Nuc) fractions revealing pYAP1 (Y357) and YAP1 protein expression in the adherent and spheroid culture of A549 cell cultures. (D) Co-immunoprecipitation of the nuclear fraction to detect the interaction between pYAP1 (Y357) and TEAD1. (E) Representative images of proximity ligation assay (PLA) illustrating protein-protein interactions between pYAP1 (Y357) and TEAD1. (F) ChIP-qPCR analysis using TEAD1 antibody to confirm protein-crosslinked genomic DNA fragments. (G) Western blot showing protein expression of ACSL1, ACSL4, DGAT1, DGAT2, LPIN2 and PNPLA3 in spheroid culture treated without or with 10 μM verteporfin (VP) for 24 h. (H and I) Assessment of TAG levels and cell viability in spheroid A549 cells treated without (CTR) or with 10 μM VP. (J) Western blot showing ACSL1, ACSL4, DGAT1, DGAT2, LPIN2 and PNPLA3 protein expression in spheroid A549 cells with YAP1 or TEAD1 knockdown. (K) Reduced TAG levels in spheroid A549 cells with YAP1 or TEAD1 knockdown. (L) Cell viability of spheroid A549 cells treated with or without YAP1 or TEAD1 knockdown, followed by treatment with H 2 O 2 (250 μM) for 24 h. Data are presented as mean ± SD (student's two-tailed unpaired t -test is employed in F and G; one-way ANOVA followed by Tukey's post-hoc test is employed in K and L). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: Human lung cancer cell line A549 was obtained from the ATCC and grown in F–12K (Corning, Manassas, VA, USA).

Techniques: RNA Sequencing, Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Proximity Ligation Assay, Protein-Protein interactions, ChIP-qPCR, Knockdown, Two Tailed Test