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: Deciphering the CAF-LCN2 axis: Key to overcoming anti-PD-L1 immunotherapy resistance in lung cancer

doi: 10.3892/ijmm.2026.5735

Figure Lengend Snippet: Impact of CAFs on lung cancer cell resistance to anti-PD-L1 immunotherapy. (A) Diagram outlining the influence of CAFs on the resistance of lung cancer cells to anti-PD-L1 immunotherapy. (B) Cell Counting Kit-8 assay measuring the sensitivity of A549 cells to anti-PD-L1 treatment. *P<0.05, **P<0.01 and ***P<0.001 vs. N-A549. (C) EdU labeling experiment evaluating the proliferative capacity of A549 cells (scale bar, 25 µm), with EdU labeled in green fluorescence and DAPI in blue fluorescence. (D) Transwell assay assessing the invasive ability of A549 cells (scale bar, 50 µm). (E) TUNEL labeling experiment detecting the apoptotic status of A549 cells (scale bar, 25 µm), with TUNEL staining in green fluorescence and DAPI staining in blue fluorescence. (F) Expression of LCN2 mRNA in A549 cells cultured with CM from NFs and CAFs. (G) Protein expression of LCN2 in A549 cells cultured with CM from NFs and CAFs. *P<0.05, **P<0.01 and ***P<0.001. All cell experiments were conducted in triplicate. One-way ANOVA with Tukey's multiple comparisons test was used for (B) and unpaired two-tailed Student's t-test was used for (C-G). LCN2, lipocalin 2; CAFs, cancer-associated fibroblasts; PD-L1, programmed death-ligand 1; NFs, normal fibroblasts; CM, conditioned medium; ns, not significant (P>0.05).

Article Snippet: In the present study, A549 lung cancer cells [cat. no. CCL-185; American Type Culture Collection (ATCC)] were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), while 293T cells (cat. no. CRL-3216; ATCC) were maintained in DMEM/F12 medium with 10% FBS.

Techniques: Cell Counting, Labeling, Fluorescence, Transwell Assay, TUNEL Assay, Staining, Expressing, Cell Culture, Two Tailed Test

Journal: International Journal of Molecular Medicine

Article Title: Deciphering the CAF-LCN2 axis: Key to overcoming anti-PD-L1 immunotherapy resistance in lung cancer

doi: 10.3892/ijmm.2026.5735

Figure Lengend Snippet: Impact of CAFs on anti-PD-L1 immunotherapy resistance in lung cancer cells via LCN2 expression. (A) Simplified flowchart depicting how CAFs influence anti-PD-L1 immunotherapy resistance in lung cancer cells through LCN2 expression. (B) Cell Counting Kit-8 assay assessing the sensitivity of A549 cells to anti-PD-L1 treatment. *P<0.05, **P<0.01 and ***P<0.001 vs. C-sh-NC. (C) EdU labeling experiment evaluating the proliferative capacity of A549 cells (scale bar, 25 µm), with EdU labeled in green fluorescence and DAPI in blue fluorescence. (D) Transwell assay investigating the invasive ability of A549 cells (scale bar, 50 µm). (E) TUNEL staining analyzing the apoptotic status of A549 cells (scale bar, 25 µm), with TUNEL staining in green fluorescence and DAPI in blue fluorescence. (F) Measurement of intracellular Fe 2+ and ROS levels (scale bar, 25 µm); *P<0.05, **P<0.01 and ***P<0.001; all cell experiments were conducted in triplicate. One-way ANOVA with Tukey's multiple comparisons test was used for (B-F). CAFs, cancer-associated fibroblasts; PD-L1, programmed death-ligand 1; ROS, reactive oxygen species; sh-, short hairpin; NC, negative control; Fe 2+ , iron; ns, not significant (P>0.05).

Article Snippet: In the present study, A549 lung cancer cells [cat. no. CCL-185; American Type Culture Collection (ATCC)] were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), while 293T cells (cat. no. CRL-3216; ATCC) were maintained in DMEM/F12 medium with 10% FBS.

Techniques: Expressing, Cell Counting, Labeling, Fluorescence, Transwell Assay, TUNEL Assay, Staining, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion causes the nucleolar stress response in MCF10A and A549 cells. A , schematic of the nucleolar stress response in human cells. Disruption of ribosome biogenesis causes accumulation of free 5S RNP which inhibits the ubiquitin ligase MDM2, leading to accumulation of TP53 and increased transcription of CDKN1A . B , TP53 stabilization after SPRR3 depletion in MCF10A cells. Representative images and quantification of TP53 Western blots after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. TP53 levels were normalized to total protein (trichloroethanol stain), then siNT. C , CDKN1A mRNA levels are elevated after SPRR3 depletion in MCF10A cells. After 72 h of siSPRR3 treatment, CDKN1A mRNA transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. These data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , siSPRR3 reduces SPRR3 mRNA levels in A549 cells. After 72 h of siSPRR3 treatment, SPRR3 transcript levels were detected by RT-qPCR. The data were normalized as in ( C ). E , siSPRR3 reduces SPRR3 protein levels in A549 cells. After 72 h of treatment with siSPRR3, SPRR3 protein levels were detected by Western blot. SPRR3 levels were normalized as in ( B ). F , SPRR3 depletion lowers 47S/45S pre-rRNA levels in A549 cells. Primers to the 5′ ETS of the 47S/45S rRNA were used to detect pre-rRNA levels after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. The data were normalized as in ( C and D ). G , TP53 stabilization after SPRR3 knockdown in A549 cells. Representative images and quantification of TP53 Western blots after 48h or 72h of treatment with siSPRR3 are shown. Protein levels were normalized as in ( B and E ). H , CDKN1A levels are elevated after SPRR3 depletion in A549 cells. After 72 h of treatment with siSPRR3, CDKN1A transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. Data were normalized as in ( C ). The mean ± SEM are shown alongside individual data points. For graphs with two conditions, data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. For graphs with more than two conditions, data were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: A549 human lung carcinoma cells (#CCL-185, American Type Culture Collection) were cultured in DMEM with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin.

Techniques: Disruption, Ubiquitin Proteomics, Western Blot, Positive Control, Staining, Quantitative RT-PCR, Comparison, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 drives AKT phosphorylation and maintains POLR1A levels. A , AKT phosphorylation at serine 473 (pAKT) is decreased after a 72h SPRR3 depletion in MCF10A cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). Blots were probed for pAKT, then stripped and re-probed for total AKT. Signal was measured in Bio-Rad Image Lab. pAKT levels were normalized to total protein and total AKT. Total AKT was normalized to total protein, ensuring no significant difference in overall AKT levels upon SPRR3 depletion. B , phosphorylated AKT (pAKT) levels are decreased after 72h SPRR3 depletion in A549 cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). C , POLR1A levels are decreased upon 72h SPRR3 depletion. Representative image of Western blotting and quantification of POLR1A levels in MCF10A cells. siPOLR1A was used as a positive control. D , summary of effects of siSPRR3 depletion that we have confirmed in both MCF10A cells and A549 cells. For all graphs in this figure, the mean ± SEM are shown alongside individual data points. Data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not significant.

Article Snippet: A549 human lung carcinoma cells (#CCL-185, American Type Culture Collection) were cultured in DMEM with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin.

Techniques: Phospho-proteomics, Staining, Western Blot, Positive Control

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: 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