Rational: Intracellular bacterial survival is a major factor causing chronic or recurrent infection, leading to the failure of both host defense and/or antibiotic treatment. However, the elimination of intracellular bacteria is challenging as they are protected from antibiotics and host immune attack. Recent studies have indicated that iron helps macrophages against intracellular bacteria, contradictory to traditional “nutritional immunity”, in which iron is considered a key nutrient for bacterial survival in host cells. However, how iron facilitates intracellular bacterial death has not been fully clarified. In this study, we found that ferroptotic stress can help macrophages suppress intracellular bacteria by reversing the importation of ferrous iron into bacterial vacuoles via ferroportin and thereby inducing in situ ferroptosis-like bacterial death.Methods: A macrophage model of bacterial invasion was established to monitor dynamic changes in ferroptotic hallmarks, including ferrous iron and lipid peroxidation. Ferroptosis inducers and inhibitors were added to the model to evaluate the relationship between ferroptotic stress and intracellular bacterial survival. We then determined the spatiotemporal distributions of ferroportin, ferrous iron, and lipid peroxidation in macrophages and intracellular bacteria. A bacterial infection mouse model was established to evaluate the therapeutic effects of drugs that regulate ferroptotic stress.Results: Ferrous iron and lipid peroxidation increased sharply in the early stage of bacterial infection in the macrophages, then decreased to normal levels in the late stage of infection. The addition of ferroptosis inducers  in macrophages promoted intracellular bacterial suppression. Further studies revealed that ferrous iron could be delivered to the intracellular bacterial compartment via inward ferroportin transportation, where ferrous iron induced ferroptosis-like death of bacteria. In addition, ferroptotic stress declined to normal levels in the late stage of infection by regulating iron-related pathways in the macrophages. Importantly, we found that enhancing ferroptotic stress with a ferroptosis inducer  successfully suppressed bacteria in the mouse infection models.Conclusions: Our study suggests that the spatiotemporal response to ferroptosis stress is an efficient pathway for macrophage defense against bacterial invasion, and targeting ferroptosis may achieve therapeutic targets for infectious diseases challenged by intracellular pathogens. Salmonella typhimurium, as FPN imports iron into Salmonella-containing vacuoles (SCV) and locally induces reactive oxygen species (ROS) via the sequential reactions of NADH oxidase 2 (Nox2) and Fenton catalysis Persistent infections due to intracellular bacteria are a serious problem in recovery. As the first line of defense against bacterial infection, macrophages have evolved versatile strategies to combat intracellular bacteria 3O4 and Fe3S4) can directly kill bacteria by releasing ferrous iron (not ferric iron) to induce lipid peroxidation in bacteria Salmonella enteritidis and Staphylococcus aureus can be suppressed by the addition of iron-containing nanomaterials or ferrous iron to macrophages. Notably, only ferrous iron, not ferric iron, exhibits antibacterial activity, consistent with the phenomenon that most bacterial siderophores chelate and deliver ferric iron as an iron nutrient resource We previously demonstrated that iron-containing nanomaterials  and lipid peroxidation, in macrophages upon bacterial invasion over time. We then assessed the correlation between ferroptotic stress with ferroptosis inducers and inhibitors and survival rate of intracellular bacteria in RAW264.7 cells and primary bone marrow protomacrophages (BMMs). We also determined the spatiotemporal distributions of iron, FPN, and lipid peroxide with intracellular bacteria. Additionally, the therapeutic efficacy of targeting ferroptotic stress regulation was assessed in established mouse models bearing bacterial infections.All animal studies were performed following the protocols approved by the Institutional Animal Care and Use Committee of Yangzhou University and Institutional Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences. All mice were discarded according to standard approved protocols after the experiments were completed.2. The cells were collected at the logarithmic growth stage, resuspended in the same complete culture medium, and inoculated (7 × 105 cells) into a 6-well plate containing 1 mL of complete culture medium (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin). Cells were then cultured at 37 °C under 5% CO2 to 80%-90% confluence, thus achieving a cell count of 1 × 106 cells/well. Single colonies of Staphylococcus aureus (ATCC 29213) (S. aureus) and Escherichia coli (CMCC (B)44102) (E. coli) in lysogeny broth (LB) on solid agar plates were randomly selected and inoculated in 5 mL of LB liquid culture medium and cultured at 37 °C for 12 h under 180 rpm rotation. The next day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium and cultured at 37℃ for 3 h under 180 rpm until OD600 reached 0.8. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted to 2 × 108 colony forming units (CFUs)/mL with 2.5 mL of RPMI 1640 culture medium. The RAW264.7 cells were then infected with 100 µL of S. aureus and E. coli for 1 h, respectively (MOI = 20:1), and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. The cells were then washed three times with phosphate-buffered saline (PBS) and cultured at 37 °C under 5% CO2 in 1 mL of RPMI 1640 containing 1% FBS for 1, 3, 6, 12 and 24 h. Iron levels , and ferric iron (Fe3+)) in the cells were measured using a Micro Serum Iron Concentration Assay Kit (Solarbio). Intracellular ferrous iron was also detected with a FeRhoNoxTM-1 fluorescent probe . Then, 200 µL of 5 µM FeRhoNoxTM-1 working solution was added to the cells, followed by 45 min of incubation at 37 °C. Intracellular ferrous fluorescence intensity was measured using a Zeiss confocal microscope (Zeiss LSM980) or flow cytometry . Total glutathione (GSH), reduced glutathione GSH, and oxidized glutathione disulfide (GSSG) were detected using GSH and GSSG Assay kits (Beyotime Biotechnology). Intracellular lipid peroxidation levels were detected with a BODIPY 581/591 C11 probe, with 200 µL of 2 µM BODIPY 581/591 C11 probe working solution added to the cells with 30 min of incubation at 37 °C. Fluorescence intensity of lipid ROS was detected by flow cytometry . Changes in mitochondrial membrane potential (MMP) were detected with a JC-1 fluorescent probe (Beyotime Biotechnology). Fluorescence of J-aggregates was measured using a multi-scan spectrum with excitation at 525 nm and emission at 590 nm. Cell activity following bacterial infection was assayed with a Cell Counting Kit-8 (CCK-8) (DOJINDO).RAW264.7 mouse macrophages were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences . The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (BI), supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Corning) for 12 h at 37 °C under 5% COS. aureus (ATCC 29213), E. coli (CMCC (B)44102), and Salmonella pullorum  on LB solid agar plates were randomly selected and inoculated in 5 mL of LB liquid culture medium and cultured at 37 °C for 12 h under 180 rpm rotation. The following day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium and cultured at 37 °C for 3 h at 180 rpm until OD600 reached 0.8. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted to 2 × 108 CFUs/mL with 2.5 mL of RPMI 1640 culture medium. Then, 100 μL of bacterial inoculation was mixed with 900 μL of RPMI 1640 containing 1% FBS as the control. Another 100 μL of bacterial inoculation was mixed with 900 μL of RPMI 1640 containing gentamicin (200 μg/mL) and 1% FBS as the experimental group. After incubation at 37℃ for 1 h and 3 h, bacterial viability was checked by plating bacteria at a proper dilution and calculating the bacterial number in CFUs/mL.Single colonies of S. aureus, E. coli, and S. pullorum at MOI = 20:1 for 1 h, respectively, with the extracellular bacteria killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 1 mL of RPMI 1640 containing 1% FBS for 0.5, 1, and 2 h. A sample of the cells was lysed using 1 mL of buffer containing 0.4% Triton X-100. The number of viable bacteria was counted by plating serial dilutions of bacteria on LB solid medium.The effect of gentamicin on intracellular bacterial activity was then examined. RAW264.7 cells were inoculated in 6-well plates containing 1 mL of complete culture solution (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin). At the logarithmic growth phase, the RAW264.7 cells were infected with S. aureus (ATCC 29213), E. coli (CMCC (B)44102), and Salmonella pullorum  as the control. Another 100 μL of bacterial inoculation was mixed with Fe2+ (100 μL)  and H2O (800 μL) as the experimental group. After incubation at 37 °C for 30 min, bacterial viability was checked by plating bacteria at a proper dilution and calculating the bacterial number in CFUs/mL. Inhibition assays with 0.4% Triton X-100 (Beyotime Biotechnology), Ras-selective lethal small molecule 3 (RSL3) (TargetMol), sulfasalazine (SSZ) (TargetMol), acetaminophen (APAP) (TargetMol), ethylenediaminetetraacetic acid (EDTA), deferoxamine mesylate (DFO) (TargetMol), ferrostatin-1 (Fer-1) (TargetMol), Fe3+, and hepcidin (Abcam) were conducted using the same system and procedure. Inhibition assays with Fe2+ in combination with EDTA or Fer-1 also used under the same system and procedure, although in addition to 100 μL of Fe2+ (12.5 µM), an additional 100 μL of EDTA or Fer-1 and 700 μL of H2O were added to maintain the system at 1 mL.Single colonies of S. aureus and E. coli on LB solid agar plates were randomly selected and inoculated in 5 mL of LB liquid medium and cultured at 37 °C for 12 h under 180 rpm rotation. The next day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium and cultured at 37 °C for 3 h at 180 rpm until OD600 reached 0.8. The bacterial precipitate (1 mL) was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted with H2O (900 μL). Then, 100 μL of Fe2+ (50 µM) was mixed with S. aureus (900 μL) and E. coli (900 μL) at 37 °C for 30 min, respectively. Morphology was examined by SEM. First, the bacterial suspension was resuspended in glutaraldehyde  for 24 h at 4 °C under dark conditions. Bacterial cells were then washed and treated with ethanol gradient dehydration , followed by drying with a critical point dryer. Finally, the bacterial cells were sputter-coated with platinum and analyzed via SEM (Hitachi-S4800) at a working voltage of 15.0 kV and working current of 10 µA under 40 K magnification Single colonies of S. aureus, E. coli, and S. pullorum incubated with Fe2+ (50 µM) were observed by TEM. Cells were fixed with 2.5% (vol/vol) glutaraldehyde with phosphate buffer (PB)  and washed four times in PB. The cells were then postfixed with 1% (wt/vol) osmium tetroxide in PB for 2 h at 4 °C and dehydrated through a graded ethanol series  in pure acetone (2 × 10 min). Samples were infiltrated in graded mixtures  of acetone and SPI-PON812 resin , and 8.9 g of N-methylolacrylamide (NMA). Finally, the cells were embedded in pure resin with 1.5% benzyldimethylamine (BDMA) and polymerized for 12 h at 45 °C and 48 h at 60 °C. Ultrathin sections (70 nm thick) were produced using a microtome (Leica EM UC6), double-stained using uranyl acetate and lead citrate, and examined using TEM (FEI Tecnai Spirit 120 kV).The internal structures of S. aureus, E. coli, and S. pullorum stimulated by ferrous iron were detected using a BODIPY 581/591 C11 fluorescent probe (Invitrogen). Single colonies of S. aureus, E. coli, and S. pullorum on LB solid agar plates were randomly selected and inoculated in 5 mL of LB liquid medium and cultured at 37 °C for 12 h under 180 rpm rotation. The next day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium and cultured at 37 °C for 3 h at 180 rpm until OD600 reached 0.8. The bacterial precipitate (1 mL) was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted with H2O (900 μL). Then, 100 μL of Fe2+  was mixed with S. aureus (900 μL), E. coli (900 μL), and S. pullorum (900 μL) at 37 °C for 30 min, respectively. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C, after which 200 µL of BODIPY 581/591 C11 probe (2 µM) working solution was added to the bacteria for 30 min of incubation at 37 °C. The lipid ROS level was measured by a multi-scan spectrum with excitation at 488 nm and emission at 525 nm.Lipid ROS levels in 2(3) probe. The S. aureus, E. coli, and S. pullorum precipitates (1 mL) at the logarithmic growth stage were obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted with H2O (900 μL). Then, 100 μL of Fe2+  was mixed with S. aureus (900 μL), E. coli (900 μL), and S. pullorum (900 μL) at 37 °C for 30 min, respectively. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C, after which 100 µL of 30 µM DIOC2(3) (US EVERBRIGHT) probe working solution was added to the bacteria for 30 min of incubation at 37 °C. Bacterial membrane potential was measured using a multi-scan spectrum with Ex/Em = 480/525 nm (green fluorescence) and Ex/Em = 530/590 nm (red fluorescence). Red/green fluorescence ratios were calculated using population mean fluorescence intensities for bacteria.Changes in bacterial membrane potential following ferrous iron treatment were detected using a DIOCS. aureus and E. coli precipitates (1 mL) at the logarithmic growth stage were obtained by centrifugation at 3 600 rpm for 5 min and diluted with H2O (450 μL). Then, 50 μL of Fe2+  was mixed with S. aureus (450 μL) and E. coli (450 μL) at 37 °C for 30 min, respectively. The supernatant was collected by centrifugation at 3 600 rpm for 5 min at 4 °C. The LDH activity in the supernatant was then measured using an LDH assay kit (Solarbio).Changes in bacterial membrane integrity following ferrous iron treatment were detected using an LDH assay kit (Solarbio). The S. aureus and E. coli precipitates (1 mL) at the logarithmic growth stage were obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted with H2O (900 μL). Then, 100 μL of Fe2+  was mixed with S. aureus (900 μL) and E. coli (900 μL) at 37 °C for 30 min, respectively. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C, after which 1 mL of 5 µM SYTO 9 (Invitrogen) working solution was added to the bacteria for 30 min of incubation at 37 °C. After washing twice with PBS, 1 mL of 10 µM propidium iodide (PI) (Beyotime Biotechnology) working solution was added to the bacteria for 30 min of incubation at 37 °C. After washing twice with PBS, DAPI (Beyotime Biotechnology) was used to stain the nuclei. After washing, live and dead bacteria after ferrous treatment were observed using a Zeiss confocal microscope (Zeiss LSM980).The 2 for 3 d, with the culture medium changed. The cells were continuously cultured until day 5, with all semi-adherent cells, i.e., enriched mouse BMMs, then collected. The BMMs were labelled with a FITC-CD 11b fluorescent probe (Invitrogen) for 30 min at 4 °C and the purity of the isolated cells was assayed by flow cytometry .Six-week-old male Balb/C mice  were sacrificed by cervical dislocation, immersed in 75% alcohol for 3 min, and then soaked in PBS containing 2% penicillin/streptomycin (Sangon Biotech) for 3 min. The femur and tibia were taken aseptically and immersed in 10 mL of RPMI 1640 medium containing 2% penicillin/streptomycin. Bone marrow was flushed into a sterile 15-mL centrifuge tube with a 1-mL syringe drawn from the 1640 medium and then centrifuged at 1 500 rpm for 10 min at 4 °C. The supernatant was discarded and 5 mL of fresh 1640 medium was added to resuspend the cells, followed by centrifugation at 1 500 rpm for 10 min at 4 °C. The supernatant was discarded, and the cells were resuspended in 10 mL of complete culture medium (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin + 20 ng/mL recombinant mouse granulocyte macrophage colony stimulating factor (rmGM-CSF)), inoculated into cell culture dishes (100 mm), and incubated in an incubator at 37 °C with 5% COS. aureus, E. coli, S. pullorum, and Salmonella enteritidis (SC070) (S. enteritidis) at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 900 µL of RPMI 1640 containing 1% FBS, with 100 µL of ferroptosis inducers , SSZ , and APAP ) or inhibitors , EDTA , and DFO ) added to the cell culture medium for 6 h (RSL3 incubated for 3 h). A sample of the cells was lysed using 1 mL of buffer containing 0.4% Triton X-100 for 30 min. The number of viable bacteria was counted by plating serial dilutions of bacteria on LB solid medium. Cell activity after bacterial infection and treatment with ferroptosis inducers and inhibitors was assayed with a Cell Counting Kit-8 (CCK-8) (DOJINDO).RAW264.7 cells were inoculated in 6-well plates containing 1 mL of complete culture solution (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin). At the logarithmic growth phase, the RAW264.7 cells were infected with S. aureus and E. coli at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 900 µL of RPMI 1640 containing 1% FBS, with 100 µL of ferroptosis inducers (Fe2+ (50µM), RSL3 (5 µM), SSZ (10 µM), and APAP (10 µM)) or inhibitors (Fer-1 (10 µM), EDTA (50 µM), and DFO (50 µM)) added to the cell culture medium for 6 h. TEM was used to observe the bacteria in the cells. Host cell morphological images with different treatments were captured using a Zeiss LSM980confocal microscope.RAW264.7 cells were infected with S. aureus, E. coli, and S. pullorum at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 900 µL of RPMI 1640 containing 1% FBS, with 100 µL of ferroptosis inducers , SSZ , and APAP ) or inhibitors  and DFO ) added to the cell culture medium at 6 h. A sample of the cells was lysed using 1 mL of buffer containing 0.4% Triton X-100. The number of viable bacteria was counted by plating serial dilutions of bacteria on LB solid medium. Cell activity after bacterial infection and treatment with ferroptosis inducers and inhibitors was assayed with a Cell Counting Kit-8 (CCK-8) (DOJINDO) and Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology).The BMMs were inoculated in 6-well plates containing 1 mL of complete culture solution (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin). At the logarithmic growth phase, RAW264.7 cells were infected with S. aureus, E. coli, and S. pullorum at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 900 µL of RPMI 1640 containing 1% FBS, with 100 µL of Fe2+  or Fe3+  then added to the cell culture medium for 6 h. A sample of the cells was lysed using 1 mL of buffer containing 0.4% Triton-X for 30 min. The number of viable bacteria was counted by plating serial dilutions of bacteria on LB solid medium. Intracellular lipid ROS levels were detected using the BODIPY581/591 C11 fluorescent probe after incubation of ferric iron with bacteria-infected macrophages.RAW264.7 cells were inoculated in 6-well plates containing 1 mL of complete culture solution (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin). At the logarithmic growth phase, RAW264.7 cells were infected with 2. Individual colonies of BL21 (E. coli strain with green fluorescence) on LB solid plates were randomly selected and inoculated in 5 mL of LB liquid medium containing kanamycin (50 µg/mL), then incubated for 12 h at 37 °C with 180 rpm rotation. The following day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium containing 50 µg/mL kanamycin and cultured at 37 °C for 3 h under 180 rpm until OD600 reached 0.8. After this, 0.5 mM isopropyl thiogalactoside (IPTG) (GenStar) was added to the bacterial solution to induce 3 h. RAW264.7 cells were infected with BL21 at MOI = 20:1 after IPTG induction for 1 h and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 0.5 mL of RPMI 1640 containing 1% FBS for 1, 3, 6, 12, and 24 h. The cells were then rinsed three times with PBS, fixed with 4% paraformaldehyde for 30 min, saturated with 0.4% Triton X-100 for 5 min, and blocked with 5% albumin bovine V (BSA) for 1 h. Cells were then incubated at 4 °C for 12 h with 200 μL of FPN primary antibodies , followed by incubation with 200 μL of fluorescein Alexa Fluor 647-conjugated goat anti-rabbit IgG  at 37 °C for 1.5 h, and staining with DAPI (Beyotime Biotechnology) for 5 min. After washing, the co-localization of FPN with BL21 was observed using a Zeiss confocal microscope (Zeiss LSM980).RAW264.7 cells at the logarithmic growth phase were collected and inoculated into 24-well cell culture plates with round coverslip (Biosharp) placed in advance, followed by the addition of 500 µL of complete culture medium. Cells were then cultured to 80% confluence at 37 °C and 5% COS. aureus and E. coli at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 1 mL of RPMI 1640 containing 1% FBS for 3 h. Co-localization of FPN with bacteria was observed by immunoelectron microscopic colloidal gold labeling. Cells were fixed with 0.1% (vol/vol) glutaraldehyde + 4% paraformaldehyde in PB  and washed four times in PB. The cell pellet was suspended in 12% wt/vol gelatin in PBS at 37 °C for 30 min, then placed on ice for 10-20 min to solidify the gelatin. The cell pellet was cut into small blocks and placed in 2.3 M sucrose overnight at 4 °C. The specimen blocks were then removed from the sucrose to aluminum specimen holders, which were frozen by immersion in liquid nitrogen. Ultrathin sections were cut at -120 °C using an ultramicrotome , and collected on grids. The grids were washed with PBS, incubated with 1% BSA for 30 min, then with anti-FPN (Thermo Fisher) primary antibodies (1:20) at 37 °C for 2 h. After washing with PBS, the grids were incubated the IgG-conjugated-gold  secondary antibodies (1:20) for 1 h at room temperature. The labeled samples were placed on uranyl acetate/methyl cellulose  drops for 5 min, then picked up by the loop and allowed to dry. The samples were then observed by TEM  at 100 kV.RAW264.7 cells were infected with 2. Individual colonies of BL21 (with green fluorescence) on LB solid plates were randomly selected and inoculated in 5 mL of LB liquid medium containing kanamycin (50 µg/mL) and incubated for 12 h at 37 °C with 180 rpm rotation. The following day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium containing 50 µg/mL kanamycin and cultured at 37 °C for 3 h under 180 rpm until OD600 reached 0.8. Then, 0.5 mM IPTG (GenStar) was added to the bacterial solution for induction for 3 h. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted to 1 × 108 CFUs/mL with 5 mL of RPMI 1640 culture medium. The RAW264.7 cells were infected with 50 µL of BL21 bacterial solution at MOI = 20:1 for 1 h, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 0.5 mL of RPMI 1640 containing 1% FBS for 6, 12, and 24 h. The cells were then washed three times with PBS. Intracellular ferrous iron was labelled with a FeRhoNoxTM-1 fluorescent probe  at 37 °C for 45 min. DAPI was used to stain the nuclei. After washing, the co-localization of ferrous iron with BL21 was observed using a Zeiss confocal microscope (Zeiss LSM980).RAW264.7 cells at the logarithmic growth phase were collected and inoculated into 24-well cell culture plates with round coverslip (Biosharp) placed in advance, followed by the addition of 500 µL of complete culture medium. Cells were then cultured to 80% confluence at 37 °C and 5% CO2. Individual colonies of BL21 (with mCherry fluorescence) on LB solid plates were randomly selected and inoculated in 5 mL of LB liquid medium containing ampicillin (50 µg/mL) and incubated for 12 h at 37 °C with 180 rpm rotation. The following day, 50 µL of the bacterial inoculation was diluted with 4 950 µL of fresh LB medium containing 50 µg/mL ampicillin and cultured at 37 °C for 3 h under 180 rpm until OD600 reached 0.8. The bacterial precipitate was obtained by centrifugation at 3 600 rpm for 5 min at 4 °C and diluted to 1 × 108 CFUs/mL with 5 mL of RPMI 1640 culture medium. The RAW264.7 cells were infected with 50 µL of BL21 bacterial solution at MOI = 20:1 for 1 h, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 0.5 mL of RPMI 1640 containing 1% FBS for 6, 12, and 24 h. Cells were washed three times with PBS. Lipid ROS were stained with 100 µL of Liperfluo (10 µM) at 37 °C for 30 min. DAPI was used to stain the nuclei. After washing, the co-localization of lipid ROS with BL21 was observed using a Zeiss confocal microscope (Zeiss LSM980).RAW264.7 cells at the logarithmic growth phase were collected and inoculated into 24-well cell culture plates with round coverslip (Biosharp) placed in advance, followed by the addition of 500 µL of complete culture medium. Cells were then cultured to 80% confluence at 37 °C and 5% COE. coli at MOI = 20:1 for 1 h, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 0.5 mL of RPMI 1640 containing 1% FBS for 1, 3, 6, 12, and 24 h. Cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, saturated with 0.4% Triton X-100 for 5 min, and blocked with 5% albumin bovine V (BSA) for 1 h. Cells were then incubated at 4 °C for 12 h with 200 μL of ferritin or transferrin receptor primary antibodies , followed by incubation with 200 μL of fluorescein Alexa Fluor 488-conjugated goat anti-rabbit IgG  at 37 °C for 1.5 h, and staining with DAPI (Beyotime Biotechnology) for 5 min. After washing, the fluorescence intensity of ferritin and transferrin receptor was observed using a Zeiss confocal microscope (Zeiss LSM980).RAW264.7 cells were inoculated in 24-well cell culture plates with round coverslip placed in advance. At the logarithmic growth phase, the RAW264.7 cells were infected with 2. After washing with ice-cold PBS, the cells were collected by centrifugation at 2 000 rpm for 5 min at 4 °C and lysed in 100 µL of RIPA buffer (Beyotime Biotechnology) containing protease inhibitor. The lysate was centrifuged at 12 000 g for 10 min at 4 °C and the protein concentrations were determined and calibrated using a BCA Protein Assay Kit (Thermo Fisher). Protein extracts were resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked for 1 h with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) and 5% skimmed milk powder. The PVDF membranes were washed three times with TBST (10 min each time). Ferroportin antibodies (Thermo Fisher) were diluted 1:1 000 in TBST  and incubated overnight at 4 °C. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies (Sigma) in a solution of TBST. Protein bands were visualized using a fully automated chemiluminescence image analysis system (Tanon-4600SF).Ferroportin shRNA  and null shRNA  were transfected into 293T cells. The viral supernatant was collected 48 h after transfection and filtered through a 0.22-μM filter. The RAW264.7 macrophages were inoculated in 6-well plates containing 1 mL of complete culture solution (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin) and infected with 1 mL of the filtered virus for 2 h, then replenished with 2 mL of fresh RPMI 1640 culture medium (RPMI 1640 + 1% FBS + 1% penicillin/streptomycin) and incubated for 24 h at 37 °C. The medium was replaced with fresh RPMI 1640 culture medium (RPMI 1640 + 1% FBS + 1% penicillin/streptomycin), followed by an additional 48 h of culture at 37 °C. After 48 h of virus infection, 2 mL of 2.5 µg/mL puromycin (GenStar) was added to the culture medium for screening. Fresh culture medium was replaced every 2 days and the cells were incubated continuously for more than a week at 37 °C under 5% COS. aureus, E. coli, and S. pullorum at MOI = 20:1 after FPN knockdown for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 1 mL of RPMI 1640 containing 1% FBS for 3 h. A sample of the cells was lysed using buffer containing 0.4% Triton X-100 for 30 min. The number of viable bacteria was counted by plating serial dilutions of bacteria on LB solid medium.The RAW264.7 cells were inoculated into a 6-well plate containing 1 mL of complete culture medium (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin). The RAW264.7 cells were infected with S. aureus, E. coli, and S. pullorum at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 1 mL of RPMI 1640 containing 1% FBS for 3 h. RAW264.7 cells cultured in 6-well plates were infected with S. aureus, E. coli, and S. pullorum at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 3 h. After washing three times with PBS, the cells were cultured at 37 °C under 5% CO2 in 1 mL of RPMI 1640 containing 1% FBS and hepcidin  for 3 h. A sample of the cells was lysed using buffer containing 0.4% Triton X-100 for 30 min. The number of viable bacteria was counted by plating serial dilutions of bacteria on LB solid medium. Immunoblot assay was used to detect the effect of hepcidin on FPN. Intracellular ferrous iron was detected with a FeRhoNoxTM-1 fluorescent probe , with 200 µL of 5 µM FeRhoNoxTM-1 working solution then added to the cells for 30 min of incubation at 37 °C. The cells were detected by flow cytometry .Hepcidin  was added to RAW264.7 cells cultured in 6-well plates for 3 h of incubation at 37 °C. The pre-treated RAW264.7 cells were then infected with S. aureus, E. coli, and S. pullorum infected at MOI = 20:1 for 1 h, respectively, and extracellular bacteria were killed with gentamicin (200 μg/mL) for 1, 3, 6, 12, and 24 h. After washing with ice-cold PBS, the cells were lysed in RIPA buffer (Beyotime Biotechnology) containing protease inhibitor. The lysate was centrifuged at 12 000 g for 10 min at 4 °C and the protein concentrations were determined and calibrated using a BCA Protein Assay Kit. Protein extracts were resolved on SDS-polyacrylamide gels, transferred to PVDF membranes, and blocked for 1 h with 0.05% TBST and 5% skimmed milk powder. The PVDF membranes were washed three times with TBST (10 min each time). Primary antibodies were diluted 1:1 000 in TBST (containing 5% BSA) and incubated overnight at 4 °C. The following primary antibodies were used: anti-HO-1 (Abcam), anti-FPN (Thermo Fisher), anti-Ferritin (Abcam), anti-NCOA-4 (Abcam), anti-Nrf2 (Abcam), anti-GPX4 (Abcam), and anti-GAPDH (Abcam). After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit (Abcam) or anti-mouse (Abcam) secondary antibodies (1:1000) in TBST for 1 h at room temperature. Protein bands were visualized using a fully automated chemiluminescence image analysis system (Tanon-4600SF).RAW264.7 cells cultured in 6-well plates were infected with 2 to 80%-90% confluence, thus achieving a cell count of 1×106. The culture was then replaced with 1 mL of RPMI 1640 containing 1%, 2.5%, and 20% FBS and incubated for 24 h at 37 °C. After washing with ice-cold PBS, the cells were lysed in RIPA buffer (Beyotime Biotechnology) containing protease inhibitor. The lysate was centrifuged at 12 000 g for 10 min at 4 °C and the protein concentrations were determined and calibrated using a BCA Protein Assay Kit. Protein extracts were resolved on SDS-polyacrylamide gels, transferred to PVDF membranes, and blocked for 1 h with 0.05% TBST and 5% skimmed milk powder. The PVDF membranes were washed three times with TBST (10 min each time). The LC3 primary antibodies (Abcam) were diluted 1:1 000 in TBST (containing 5% BSA) and incubated overnight at 4 °C. After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit (Abcam) secondary antibodies (1:1000) in TBST for 1 h at room temperature. Protein bands were visualized using a fully automated chemiluminescence image analysis system (Tanon-4600SF).RAW264.7 cells were inoculated into a 6-well plate containing 1 mL of complete culture medium (RPMI 1640 + 20% FBS + 1% penicillin/streptomycin). Cells were then cultured at 37 °C under 5% COin vivo infection models. The mice (five in each group) were infected in the tail vein with S. aureus (1 × 106 CFUs) or S. pullorum (1 × 106 CFUs) at the logarithmic growth stage. After 12 h of infection, blood was collected from the orbital vein and erythrocytes were lysed in erythrocyte lysate (1 mL) (TIANGEN) at room temperature. The mixture was centrifuged at 3 500 rpm for 10 min at 4 °C to remove the erythrocyte lysate. The cells were then divided equally into two portions. One portion of cells was labelled with 200 µL of BODIPY 581/591 C11 (2 µM), followed by incubation for 30 min at 37 °C. After washing with PBS, 100 µL of APC CD 11b antibody (Invitrogen) working solution (1:1 000) was added to the cells, followed by 30 min of incubation at 4 °C. The cells were then washed three times with PBS, and changes in lipid ROS in peripheral blood macrophages were detected by flow cytometry.Six-week-old male Balb/C mice  were used to establish TM-1 probes (5 µM), followed by incubation for 45 min at 37 °C. After washing with PBS, 100 µL of APC CD 11b antibody (Invitrogen) working solution (1:1 000) was added to the cells, followed by 30 min of incubation at 4 °C. The cells were then washed three times with PBS, and changes in ferrous iron in peripheral blood macrophages were detected by flow cytometry.The other cells were labelled with 200 µL of FeRhoNoxAfter 12 h of infection, 100 μL of SSZ  or DFO  was administered intravenously twice a day, with a total of five treatments per mouse. Control mice with bacterial infection received 100 μL of PBS only via intravenous injection. Body weight and behavior, including feeding, drinking, and activity, were continuously observed and recorded for 3 days. All mice were sacrificed on day 3 after infection, and their hearts, livers, lungs, spleens, and kidneys were collected in 5 mL of PBS. Tissue samples were ground under aseptic conditions. Viability of bacteria was assessed by calculating bacterial number (CFUs/mouse) in organs. Antibacterial efficiency was determined by calculating the ratio of bacterial number between the SSZ or DFO-treated groups and PBS-treated group.After 12 h of infection, 100 μL of SSZ (100 µM) or DFO (100 µM) was administered intravenously twice a day, with a total of five treatments per mouse. Control mice with bacterial infection received 100 μL of PBS only via intravenous injection. All mice were sacrificed on day 3 after infection. Their main organs  were collected in 10 mL of 4% paraformaldehyde, then fixed in paraffin and stained with hematoxylin-eosin (H&E) to examine changes in organ histomorphology.TM-1 probes (5 µM), followed by incubation for 45 min at 37 °C. After washing with PBS, 100 µL of APC CD 11b antibody (Invitrogen) working solution (1:1 000) was added to the cells, followed by 30 min of incubation at 4 °C. The cells were then washed three times with PBS, and changes in ferrous iron in peripheral blood macrophages were detected by flow cytometry.After 12 h of infection, 100 μL of SSZ (100 µM) or DFO (100 µM) was administered intravenously twice a day, with a total of five treatments per mouse. Control mice with bacterial infection received 100 μL of PBS only via intravenous injection. All mice were sacrificed on day 3 after infection, and blood was collected from the orbital vein and erythrocytes were lysed in erythrocyte lysate (1 mL) (TIANGEN) at room temperature. The mixture was centrifuged at 3 500 rpm for 10 min at 4 °C to remove the erythrocyte lysate. The cells were then divided equally into two portions. One portion of cells was labelled with 200 µL of BODIPY 581/591 C11 (2 µM), followed by incubation for 30 min at 37 °C. After washing with PBS, 100 µL of APC CD 11b antibody (Invitrogen) working solution (1:1 000) was added to the cells, followed by 30 min of incubation at 4 °C. The cells were then washed three times with PBS, and changes in lipid ROS in peripheral blood macrophages were detected by flow cytometry. The other cells were labelled with 200 µL of FeRhoNoxin vivo infection models. Mouse BMMs were infected with S. aureus or S. pullorum following the in vitro intracellular bacterial model. Following infection, cells were collected from a single well of a 6-well plate, before being resuspended in 1 mL of PBS, after which 100 μL (105 cells) of the mixture was intravenously injected into each mouse. After 12 h of infection, 100 μL of SSZ (100 µM) or DFO (100 µM) was administered intravenously twice a day, with a total of five treatments per mouse. Control mice with bacterial infection received 100 μL of PBS only via intravenous injection. Body weight and behavior, including feeding, drinking, and activity, were continuously observed and recorded for 3 days. All mice were sacrificed on day 3 after infection, and kidneys were collected in 5 mL of PBS. Tissue samples were ground under aseptic conditions. Viability of bacteria was assessed by calculating bacterial number (CFUs/mouse) in the two kidneys. Antibacterial efficiency was determined by calculating the ratio of bacterial number between the SSZ or DFO-treated groups and PBS-treated group.Six-week-old male Balb/C mice  were used to establish in vivo infection models. Fifteen healthy Balb/C mice were randomly divided into three groups (five in each group). Two groups were intravenously injected with 100 μL of SSZ (100 µM) and DFO (100 µM) twice a day, respectively. One group was intravenously injected with PBS as the control. On day 3, the injections were stopped, and the mice were housed until day 7. Body weight and behavior, including feeding, drinking, and activity, were continuously observed and recorded for 7 days. All mice were sacrificed on day 7 after infection and blood was collected from the orbital vein to assess the levels of hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), red blood cells (RBC), hematocrit (HCT), white blood cells (WBC), platelet count (PLT), neutrophilic granulocytes (NEUT). The main organs, i.e., liver, kidneys, spleen, lungs, and heart, were collected in 10 mL of 4% paraformaldehyde, then fixed in paraffin and stained with H&E to examine changes in organ histomorphology.Six-week-old male Balb/C mice were used to establish  t-test for column analyses. For comparing multiple groups, we used two-way analysis of variance (ANOVA). NS, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001, and ****P < 0.0001.All experiments were performed as biological replicates. Sample size for each experimental group per condition is reported in the corresponding figure legends and Methods section. For bacterial and cell experiments, sample size was not predetermined, and all samples were included in the analysis. In the animal experiments, no statistical methods were used to predetermine sample size (n = number of mice per group), and all animals were used for analysis unless they died. GraphPad Prism v7.0 (GraphPad Software) was used for statistical analyses. For comparing two groups, we used two-tailed Student'sS. aureus or E. coli, then treated with gentamycin (200 µg/mL) for 3 h to kill the extracellular bacteria  inhibited more than 90% of S. aureus, 90% of E. coli, 50% of S. pullorum, and 60% of S. enteritidis in the RAW264.7 cells . These results indicate that ferroptosis inducers and inhibitors affect bacterial viability mainly through ferroptotic processes rather than direct inhibition of bacteria. Importantly, our results indicate that transient ferroptosis activity can assist macrophages to suppress invading bacteria, with inhibitory efficiency in the order: E. coli > S. aureus > S. pullorum.Ferroptosis inducers, including SSZ y Figure I-J. Simis Figure K-N. Ferrs Figure O. When D.7 cells P-S. Thes.7 cells . To confs Figure A-B. In cy Figure A-B. Comp E. coli A and C.  showed significant bacterial inhibition in the BMMs, reducing S. aureus, E. coli, and S. pullorum by more than 60%, 85%, and 30%, respectively  was first engineered with green fluorescence and then used in a RAW264.7 cell infection model, with FPN tested via immunofluorescence imaging. As shown in Figure The above results indicated that cellular ferroptotic stress leads to suppression of intracellular bacteria. However, bacteria can be found in phagosomes via phagocytosis or in late phagolysosomes via fusion of phagosomes and lysosomes. This raises an interesting question, namely how are ferroptotic signals transmitted to compartmentalized bacteria? We hypothesize that the key factor in ferroptotic stress, i.e., ferrous iron, may be transported to bacterial vesicles. FPN is a major transporter that exports ferrous iron from the cell to extracellular compartments in macrophages S. aureus, E. coli, and S. pullorum in the infected macrophages. Moreover, the higher the efficiency of FPN knockdown, the more bacteria were counted in the macrophages  treatment led to a significant reduction in intracellular bacteria in the RAW264.7 cells, i.e., 96% for S. aureus, 91% for E. coli, and 94% for S. pullorum  ( E. coli genes (1.9% down-regulated and 8.2% up-regulated) (2(3) fluorescent probe revealed that ferrous iron (200 µM) decreased the membrane potential of S. aureus, E. coli, and S. pullorum by 39%, 31%, and 30%, respectively  A, which gulated) B. Similagulated) A, which gulated) B. Detecty Figure D. Bactery Figure E-F. In ay Figure . Compariactivity G-I. Thusactivity J-L. Impoactivity M-O. ThesTM-1 was used as a probe of intracellular ferrous iron. Fluorescent imaging showed that the intracellular fluorescence intensity of ferrous iron increased considerably in the whole cell, and the BL21 bacteria were located in the area of high ferrous probe density in the early stage of infection (post 6-12 h). In contrast, at 24 h post infection, all bacteria inside the cells disappeared and the fluorescence intensity of ferrous iron decreased to the level found in the control cells , which demonstrated that intracellular lipid ROS increased and co-localized with BL21 when infected cells were cultured for 6-12 h. Similar to the dynamic changes in ferrous iron, all bacteria inside the cells were killed and the fluorescence intensity of lipid ROS was reduced after 24 h Figure G. Surpriectively , indicat3+, and combines with the membrane protein Tf receptor 1 (TfR1), which enters the nuclear endosome under endocytosis. Fe3+ is then reduced to Fe2+ by six-transmembrane epithelial antigen of prostate 3 (STEAP3), which is then stored in the unstable iron pool and ferritin E. coli bacteria were used in a RAW264.7 cell infection model via immunofluorescence imaging. Results showed no significant change in the fluorescence intensity of TfRs in the whole cells after bacterial infection  is present on the cell membrane and mediates the transportation of iron from the extracellular to intracellular compartment. Ferric iron binds to transferrin (Tf) on the cell membrane to form Tf-Fenfection . Thus, tnfection , indicatIn addition to generating ferrous iron, retaining ferrous iron inside the cell is also important. As such, we analyzed changes in FPN in the RAW264.7 cells during bacterial infection by western blotting. As expected, FPN abundance decreased in the RAW264.7 cells at 3-12 h after bacterial infection, and then increased at 24 h Figure .in vivo bacterial infection can be treated by ferroptosis regulators. Firstly, lipid ROS levels in peripheral blood macrophages were measured in mice after intravenous infection by S. aureus or S. pullorum in the tail vein. Peripheral blood was taken from mice 24 h after infection and peripheral blood macrophages were collected for labelling with APC CD 11b antibodies and BODIPY 581/591 C11 probes, respectively  or DFO , respectively. By analyzing bacterial viability in the hearts, livers, spleens, lungs, and kidneys of mice, the highest inhibition efficiency was achieved after treatment with 100 µM SSZ or DFO (S. aureus and S. pullorum (at the logarithmic growth stage) via the tail vein, respectively. After 12 h of infection, 100 μL of SSZ  or 100 μL of DFO  was administered intravenously twice a day, with a total of five treatments per mouse , liver, spleen, and lungs of the mice, the SSZ-treated group exhibited a reduction in S. aureus and S. pullorum compared to those mice treated with PBS alone , central venous congestion (arrow), and massive inflammatory cell infiltration (long arrow). These symptoms disappeared after SSZ treatment. The liver structure of S. pullorum-infected mice was destroyed, with hepatocyte necrosis (short arrow) and central venous congestion (arrow). SSZ treatment reduced central venous congestion. Mature and immature megakaryocytes were increased in the spleen of S. aureus-infected mice but decreased after SSZ treatment and increased after DFO treatment. The same changes were observed after S. pullorum infection. There were no significant pathological changes in the lungs of S. aureus-infected mice, and no significant pathological changes in the lungs after SSZ treatment. However, the lungs of DFO-treated mice showed alveolar cavity hemorrhage (arrow). Kidney sections from S. aureus-infected mice exhibited renal cell necrosis (long arrow), which was reduced by SSZ treatment. In contrast, the area of necrosis was not reduced after DFO treatment (long arrow) and inflammatory cell infiltration foci appeared (short arrow). In summary, the liver, kidney, and spleen of bacteria-infected mice showed varying degrees of pathological changes. The lesions were reduced after SSZ treatment but aggravated after DFO treatment.Furthermore, the heart, liver, spleen, lungs, and kidneys of bacteria-infected mice were taken for H&E staining to analyze pathological changes. As shown in in vivo bacterial infection model with macrophages. As shown in Figure S. aureus or S. pullorum, followed by gentamycin treatment to kill extracellular bacteria while retaining viable bacteria inside the cell. The BMMs (105 cells/mouse) were intravenously injected into Balb/C mice. After 12 h of infection, 100 μL of SSZ (100 µM) or 100 μL of DFO (100 µM) was administered intravenously twice a day, with a total of five treatments per mouse  and DFO (100 µM) in healthy Balb/c mice and examined their influence on body weight, complete blood count, and major organs. Results showed that SSZ and DFO administration did not affect body weight  in macrophages, in which the hepcidin-FPN axis transports iron from the cytosol to SCV and generates ROS via Fenton reaction with hydrogen peroxide produced by NADPH oxidase (NOX) In the current study, we identified a general antibacterial strategy in macrophages, which transmits the cytosol ferroptosis messenger (ferrous iron) into intracellular bacterial compartments via FPN to induce ferroptosis-like bacterial death. Ferroptosis is a recently discovered type of programmed cell death with the hallmark of ferrous iron-dependent lipid peroxidation in vivo  of ferrous iron or ferroptosis inducers  increased lipid ROS levels and helped in macrophage defense against intracellular bacteria. In contrast, ferroptosis inhibitors, which can chelate ferrous iron  or reduce lipid ROS (Fer-1), demonstrated adverse effects. Thus, regulating ferroptosis activity in macrophages may be an effective strategy against intracellular bacteria at the early stage of infection. Furthermore, administration of the ferroptosis inducer SSZ in infected mice at 72 h significantly suppressed bacterial viability in vivo A-D by main vivo . These pin vivo , indicatE. coli was eliminated in macrophages after 24 h of infection, followed by S. aureus. In contrast, S. pullorum was difficult to kill under intracellular conditions, consistent with previous studies suggesting that Salmonella species are persistent pathogens in host cells Through FPN-mediated inward iron transportation, ferroptotic damage was successfully transmitted to compartmentalized bacteria. However, ferroptotic stress in the macrophages was terminated in the late stage after 12 h) of bacterial invasion. The hallmarks of ferroptotic stress, including iron concentration, lipid ROS level, and mitochondrial potential, recovered at 24 h post-infection (Figure 2 h of baOverall, our findings suggest that ferroptotic stress is beneficial for macrophages against intracellular bacteria, as the stress can be transferred from the cytosol to vacuole containing intracellular bacteria via FPN transportation. This mechanism ensures effective suppression of intracellular bacteria but is harmless to the macrophages. Therefore, targeting ferroptotic stress may be a potential strategy to treat infections with intracellular pathogens.Supplementary methods and figures.Click here for additional data file.Supplementary video.Click here for additional data file.


N,N-di-p-tolylbenzo[c]phenanthrene-4-amine (3-B(Mes)2-[4]helix-9-N(p-Tol)2) 1 and 13-(dimesitylboryl)-N,N-di-p-tolyldibenzophenanthrene-8-amine (3-B(Mes)2-[5]helix-12-N(p-Tol)2) 2 gives rise to monoanions with extensive delocalization over the annulated helicene rings and the boron pz orbital. Two-electron chemical reduction of 1 and 2 produces open-shell biradicaloid dianions with temperature-dependent population of the triplet states due to small singlet-triplet gaps. These results have been confirmed by single-crystal X-ray diffraction, EPR and UV/vis-NIR spectroscopy, and DFT calculations.One-electron chemical reduction of 10-(dimesitylboryl)- Stepwise chemical reduction of D–π–A triarylborane-based helicenes gives the corresponding monoanions and dianions with delocalized unpaired electrons. The structures were confirmed by single crystal X-ray diffraction. Isolation of stable boron-containing radicals is important both in fundamental chemistry and in practical applications, such as chemical sensors, organic synthesis and polymerizations.2 Consequently, investigation of the spin density distribution associated with borane radicals has attracted increasing interest.2 Stabilized by delocalization of the unpaired electron on two or more boron atoms, a number of radicals of diboryl compounds and polyborane clusters have been isolated.4 However, mononuclear boron radicals are still relatively rare. Reduction of triarylboranes has been studied since the 1920s,5 in systems in which the boron radical was stabilized by steric shielding to prevent bimolecular reactivity or coupled with spin delocalization via π-conjugation.Three-coordinate organoboron compounds have attracted much attention for various optical and optoelectronic applications.6 Helicenes are ortho-fused polycyclic aromatic hydrocarbons with nonplanar distorted molecular orbitals.7 Spin-delocalization through helical backbones adds an interesting element of chirality. The unique inherent optical and magnetic properties resulting from unpaired electron spin delocalization within helical π-systems are of fundamental interest to the design and development of organic optoelectronic materials and devices.Spin-delocalization is an inherent feature of π-conjugated open-shell molecules containing one or more unpaired electrons.2-[4]helix-9-N(p-Tol)2) 1 and (3-B(Mes)2-[5]helix-12-N(p-Tol)2) 2.8 The donor- and acceptor-substituted nonplanar aromatic rigid helicene derivatives exhibit moderate to high fluorescence quantum yields in solution, and up to 1.00 in the solid state, which are higher than those reported for the unsubstituted helicenes.9 Such D–π–A compounds possess large electric dipole moments in the excited state, exhibiting strong intramolecular charge-transfer (ICT) emission, which make them attractive for various applications.1 Zhao and co-workers also disclosed a triarylborane-based [5]helicene with enhancement of the fluorescence efficiency or full-color circularly polarized luminescence.11 Herein, we report the stepwise chemical reduction of the D-helix-A compounds 1 and 2 to produce their corresponding monoanions and dianions. The structures of products were confirmed by single crystal X-ray diffraction. We also investigated the reduced monoanion and dianion species by EPR spectroscopy and theoretical calculations.Recently, we reported the synthesis, photophysical, and electronic properties of triarylborane-based helical donor and acceptor compounds (3-B(Mes)1 and 2, which revealed two reversible reductions at −2.29, −2.72 V (vs. Fc/Fc+) and −2.29, −2.71 V (vs. Fc/Fc+), respectively, in THF with [nBu4N][PF6] as the supporting electrolyte.8 The appearance of two reversible reduction peaks suggested that their singly and doubly reduced species might be stable enough for isolation. We therefore carried out the stepwise chemical reduction of 1 and 2 to isolate their respective monoanions and dianions, and to study their structural and electronic properties. Compounds 1 and 2 also show similar reversible oxidations, indicating that the oxidized species might also be stable enough for isolation. However, we could not isolate the radical cations of either 1 or 2, possibly due to subsequent reaction of the triarylamine radical, as observed in earlier studies.12We previously reported cyclic voltammetry studies of 1 and 2 with 1.0 equiv. of [K(18-crown-6)(THF)2] naphthalenide (3) in THF at room temperature afforded dark red solutions. Upon slow diffusion of dry n-pentane into the reaction mixture, dark red crystals of the radical monoanion salts 1·K1 and 2·K1 were obtained in 76% and 70% yields, respectively. Upon addition of 2.5 equiv. of 3 in THF, the color of the solution changed to dark blue. By slow diffusion of dry n-pentane into the reaction mixture, dark blue crystals of the dianion salt 1·K2 and a disordered 1 : 1 co-crystal of 2·K1 and 2·K2 were obtained in 60% and 68% yields, respectively 2 group is disordered and one of the K(18-crown-6) moieties lies on an inversion center and is completely disordered via inversion symmetry  moieties per molecule. It is most likely that the monoanion and dianion have very similar molecular configurations and that the influence of the second reduction on the structural parameters is relatively small. However, the presence of slightly different geometries is reflected in enlarged anisotropic displacement parameters and in the disorder of the B(Mes)2 moiety; thus, it is not possible to comment further on bond distances within the monoanionic and dianionic helicene units.The structures of the monoanions etry see . The pot1·K1, no interaction is observed between the potassium cation and the helicene core. Instead, the potassium ion is coordinated by 18-crown-6 and two THF molecules –helicene–K(18-crown-6)–helicene–K(18-crown-6)–THF sequence –3.461(4) Å in 2·K1/2·K2  bond is significantly shortened by −0.024 to −0.035 (3) Å with respect to their neutral state, and possibly further for 2·K1/2·K2, but the esds for the latter structure do not allow an accurate analysis (Table S2via the newly populated MOs (vide infra). As a result, the B–C(mesitylene) bonds are slightly lengthened by ca. +0.012 to +0.026 (2–4) Å in the radical monoanions. In the monoanion 1·K1, the torsion angle between the BC3 plane and the helical terminal ring P1 (4.93(7)°) is significantly smaller than that in the neutral compound 1 (24.30(6)°). In the reduced anions 2·K1 and 2·K1/2·K2, torsion angles are similar to those in the neutral species 2. However, as noted above, interaction is observed between the potassium ion and the helicene core next to boron (P1 and P7). These results indicate delocalization of the single electron between boron and the helicene core. With respect to the NC3 plane, a decrease of the sum of C–N–C angles is observed from 359.40(14)° and 359.98(11)° in the neutral compounds 1 and 2, respectively, to 355.51(16)° and 355.5(2)° in the reduced compounds . In contrast to the intense emission from solutions of neutral compounds 1 and 2,8 there was no detectable emission from the monoanions and dianions in the NIR region.The UV/vis-NIR absorption spectra of er argon . The low1 and 2, which is in agreement with the observation that some triarylamines tend to form unstable short-lived radicals,12 we were able to investigate the properties of the radical cations by performing UV/vis-NIR spectroelectrochemical measurements in CH2Cl2/0.1 M [nBu4N][PF6] as depicted in 1 and 2 occur at 1071 and 972 nm, respectively.Even though we were not successful in isolating the products of the chemical oxidation of compounds 1·K1 and 2·K1 were measured in THF at room temperature  = 4.5 G for 1·K1 and giso = 2.003, a(11B) = 4.2 G for 2·K1, respectively) which are smaller than those of other triarylborane radical monoanions, such as [Ph3B]− (7.84 G)5 and [Mes3B]− (9.87 G).13 The relatively low values for the boron hyperfine couplings indicate that the spin density is delocalized onto the annulated rings of the respective helicenes in 1·K1 and 2·K1, which is in agreement with the calculated spin densities of these monoanions, vide infra. A good fit to the experimental data is obtained by considering couplings to the three protons of the adjacent benzene ring of the helicene.EPR spectra of the radical monoanions of perature . These s1·K2 and 2·K2 were recorded in THF  basis set.15 Spin density plots  transitions at 1117 and 917 nm . While the major contribution to the first excited state is again attributed to a SOMO → LUMO transition, the second excited state involves contributions from LUMO+1 to LUMO+4, with both states characterized as LE states.In order to investigate the electronic structure of ty plots  of 11− a Table S3, respect Table S3, which l1·K2 and 2·K2 without using model systems and, in addition, some of us found that the single-reference broken-symmetry DFT approaches are often sufficiently accurate.16 Thus, calculations for dianions 1·K2 and 2·K2 (as the naked species 12− and 22−) were carried out using the UM062X functional and the 6-31++G(d) basis set in combination with the SMD variation of PCM.17 The HOMO and LUMO in the unrestricted (UM062X) method were allowed to mix in order to destroy α–β and spatial symmetries. Within this approximation, an open-shell biradical S0 state would be described as a mixture of the singlet and triplet states, as indicated by an expectation value (〈S2〉) of about 1.0. If the energy difference between the orbitals is sufficiently small but not zero, the 〈S2〉 value is between one and zero, and the system can be described as “biradicaloid”.18 The energy splitting arises from possible interactions between both radical centers. For the dianions 1·K2 and 2·K2, biradicaloid S0 open-shell systems are found to be the most stable electron configurations , but the relative stability of open-shell S0 remains the same  of 1·K2 and 2·K2 as open-shell singlet and triplet with and without SMD are shown in Fig. S14–S21.z)–aryl(helicene) interaction in the FMOs of the open-shell singlet structures, which leads to a shortening of the B–C bond lengths of −0.013 to −0.018 Å with respect to their neutral states (Tables S4 and S52·K1/2·K2 (vide supra). The energy difference between the two highest α and β orbitals (ΔEαβ) is 31.36 kJ mol−1 for 1·K2 and 23.35 kJ mol−1 for 2·K2, respectively. However, larger splittings are found for the gas phase calculation (ΔEαβ = 39.85 and 37.92 kJ mol−1 for 1·K2 and 2·K2), in which we observe significant demixing between open-shell and closed-shell singlet states as indicated by the larger 〈S2〉 values in the gas phase.A multireference approach such as NEVPT2 is able to describe open-shell biradical structures very accurately. However, it is too demanding for the structures urations . The sma Table S6. The calvide supra). The overestimation of the singlet-triplet energy gap might result because the influence of the counter-ion is neglected.19 This was probed by a single point calculation on the experimental geometrical parameters obtained for 2·K1/2·K2, i.e., in the presence of the counterions. While 〈S2〉 drops to 0.1537 indicating significant demixing, the vertical singlet triplet gap decreases to 18 kJ mol−1. Thus, it is feasible that the adiabatic gap is also further decreased. Additionally, due to the delocalization error of DFT functionals,20 the interaction between both radical centers might be overestimated. Computations to investigate the influence of both error sources are under way.In general, the energy difference between closed- and open-shell singlet and triplet states in these systems is small, which is in qualitative agreement with the EPR spectra  were investigated by UV/vis-NIR absorption, EPR spectroscopy, and theoretical calculations. DFT calculations and the EPR analysis clearly revealed that the unpaired electron in both radical anions is delocalized over the annulated helicene rings and the boron pz orbital. This is supported by changes observed in the crystal structures on reduction, e.g., the B–C(helicene) bond shortening. The broken-symmetry DFT approach indicates that both dianions 1·K2 and 2·K2 can best be described as biradicaloid systems with significant admixing of the closed-shell S0 state. In addition, the properties of the radical cations of 1 and 2 were investigated by spectroelectrochemistry. Our results provide insights into helicene-based redox-active systems which may prove useful in the design of new helicenes for applications in materials chemistry.We have reduced triarylborane-based helical donor–acceptor compounds xyz coordinates for the calculated structures, and spectroscopic data can be found in the ESI.‡All experimental procedures, computational data, X. J. and Z. W. performed the experiments; J. N. and F. F. did the DFT calculations supervised by B. E.; A. F., Z. W. and J. K. carried out X-ray crystallographic studies; I. K. carried out EPR spectroscopy supervised by H. B.; M. M. carried out spectroelectrochemical measurements supervised by C. L.; T. B. M. supervised the overall project; all authors were involved in the preparation of the manuscript.The authors declare no conflict of interest.SC-012-D1SC02409D-s001SC-012-D1SC02409D-s002


The microscopic features, oxidative (isoprostane) and inflammatory markers  for control and treated cells were compared. Our results showed that 24 h of H2O2 and MIA caused oxidative stress and a concentration-dependent reduction of SW1353 cell viability without TNF-α level upregulation. H2O2 primarily induced chondrocyte apoptosis with the detection of blebbing formation, cell shrinkage and cellular debris. MIA induced S-phase arrest on chondrocytes with a reduced number of attached cells but without significant cell death. On the other hand, 24 h of IL-1β did not affect the cell morphology and viability of SW1353 cells, with a significant increase in intracellular TNF-α levels without inducing oxidative stress. In conclusion, each OA inducer exerts differential effects on SW1353 chondrocyte cell fate. IL-1β is suitable in the inflammatory study but not for chondrocyte cell death. H2O2 and MIA are suitable for inducing chondrocyte cell death and growth arrest, respectively.Osteoarthritis (OA) is the most common degenerative joint disease characterised by chondrocyte cell death. An in vitro model of chondrocyte cell death may facilitate drug discovery in OA management. In this study, the cytotoxicity and mode of cell death of SW1353 chondrocytes treated with 24 h of OA inducers, including interleukin-1β (IL-1β), hydrogen peroxide (H Osteoarthritis (OA) is a degenerative joint disease commonly found among the elderly . The exaSeveral OA models are currently used in OA research, including chondrocyte cell lines, primary articular chondrocytes and OA animals ,11,12. PChondrosarcoma cell line SW1353 is one of the most common and established cell lines used as in vitro OA model ,16,17,232O2), a non-radical ROS, has been widely reported [2O2 is membrane-permeable [2O2 has been reported in primary articular chondrocytes from animals and humans [2O2 and ROS inducers like menadione are commonly used in the in vitro approach to induce oxidative stress in chondrocytes [2O2 is rarely used but can possibly induce OA in animals [Inflammation- or age-related oxidative stress is crucial in OA progression, where chondrocytes can express the catabolic profile upon oxidative damage . The oxireported . H2O2 isermeable , wherebyermeable . The cytd humans ,35, humad humans  and SW13d humans ,37,38,39d humans . Exogenodrocytes ,42. Addi animals . Compara animals ,43.Articular chondrocytes acquire oxygen and nutrients from synovial fluid via diffusion due to the anatomically avascular and alymphatic structures of the cartilage tissues ,8,31. Th2O2 and MIA in SW1353 chondrocyte models, no direct comparison among these inducers of SW1353 cell death has been made. This research gap will impede the selection of an appropriate inducer in in vitro OA studies. So far, only a study by Martin et al. comparing the differential catabolic effects of IL-1β and H2O2 on juvenile bovine chondrocytes is available [2O2- and MIA-induced cytotoxicity and mode of cell death in SW1353 chondrocytes. The involvement of oxidative stress and inflammation were also investigated. This is the first study to investigate the differential effects of these OA inducers. Such information would benefit researchers in selecting an appropriate agent for their in vitro OA models.SW1353 chondrocytes are among the most established cell lines in in vitro OA studies. Despite the prevalent use of IL-1β, Hvailable . The dif2O2 solution was purchased from Merck Millipore  and kept at 4 °C until treatment. During treatment, the 30% H2O2 solution was pre-diluted with sterile phosphate-buffered saline (PBS) in dark conditions to prepare a 20 mM H2O2 solution, then continued to the second dilution step in culture media to prepare the treatment solution accordingly. Lyophilised recombinant human IL-1β with albumin  was purchased from Cell Signaling Technology, Inc.  and reconstituted according to the manufacturer’s instructions. Briefly, 10 μg of IL-1β powder was dissolved with sterile PBS as a 50 μg/mL stock solution at room temperature with 30 min of solubilization and occasional gentle vortexing. The dissolved IL-1β was aliquoted and kept at −80 °C until treatment. The stock solutions of IL-1β, H2O2 and MIA were always protected from light during preparation, storage and treatment. Other chemicals were purchased from Sigma-Aldrich unless stated otherwise.MIA was purchased from Sigma-Aldrich , dissolved in dimethyl sulfoxide (DMSO) as a 100 mM stock solution, aliquoted and kept at −80 °C until treatment. Concentrated 30% HSW1353 chondrocytes were purchased from ATCC  and cultured with high glucose Dulbecco’s modified Eagle’s medium (DMEM) with 4 mM l-glutamine, 10% fetal bovine serum  and 1% penicillin–streptomycin antibiotic solution  at 37 °C and 5% carbon dioxide conditions. These cells were sub-cultured every 2–3 days to maintain optimal cell growth. The cells within the first 3 to 10 passages after being revived from cryopreservation were used in the experiment.2O2 and MIA on SW1353 chondrocytes as previously described, with modifications [4 cells/mL of SW1353 chondrocytes was seeded in a 96-well plate with complete media for 24 h. On the next day, all the media were then replaced with 100 µL of complete media with a series of concentration of either IL-1β, H2O2 and MIA for another 24 h. DMSO (0.05%) was used as vehicle control (VC). At the end of the treatment, 20 µL of MTS reagent was added to each well and the plate was further incubated for another 1 h at 37 °C. The absorbance of each well was then measured at 490 nm using a Multiskan GO microplate reader . The viability of cells was calculated as a percentage with the optical density (OD) value of the VC as the denominator. The values of the 25% and 50% maximal inhibitory concentration (IC25 and IC50) were determined for subsequent experiments.CellTiter 96 AQueous One solution  containing 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) was used to determine the cytotoxicity of IL-1β, Hications ,49. Brie4 cells/mL) was seeded in a 6-well plate for 24 h, followed by treatment with H2O2 and MIA at their IC25 and IC50 values, or IL-1β at 50 and 100 ng/mL for 24 h. Before collecting the cells, the microscopic images of cells were captured at 200× magnification using an Olympus CKX31 inverted microscope with an X-CAM alpha camera and DigiAcquis 2.0 acquisition software  for morphological comparisons. All the treated cells, including the floated cells, were then collected, trypsinised and washed twice with ice-cold PBS at 200 g for 5 min. The cells were resuspended in 350 µL of Annexin V staining buffer  and stained with 5 µL of Annexin V conjugated with fluorescein isothiocyanate (Annexin V–FITC)  on ice in dark conditions for 15 min. A 10 µL solution of 50 µg/mL propidium iodide (PI) was then added to the cells and incubated for another 5 min. After the staining, another 150 µL of Annexin V staining buffer was added to the cells, and the entire content was transferred to a flow tube for analysis. At least 10,000 stained cells were recorded and analysed using a BD FACSVerse flow cytometer . The cell population with negative staining for both markers represents the viable cell population, positive staining for Annexin V–FITC represents the early apoptotic population, positive staining for PI represents the necrotic population, whereas positive staining for both markers represents a late apoptotic population. Both early and late apoptotic populations are collectively reported as the apoptotic population in this study.The mode of cell death, either via apoptosis or necrosis, was determined according to the plasma membrane externalization of phosphatidylserine and its integrity . This as4 cells/mL) was seeded in a 6-well plate for 24 h, followed by treatment with the IC50 of MIA for 24 h. A serum-free condition was used as a cell cycle arrest control. All the treated cells, including the floating cells, were then collected, trypsinised and washed twice with ice-cold PBS at 200 g for 5 min. The cells were then counted, and 1 × 105 cells were fixed with 1 mL of 70% ethanol drop by drop while being vortexed. After that, they were kept overnight. The fixed cells were washed again with ice-cold PBS at 400 g for 10 min. Subsequently, the cells were stained with 500 µL of PI/RNase staining solution  for 15 min at room temperature. At least 12,000 stained cells were recorded, and the cell cycle distribution was further analysed using ModFit LT software  via Gaussian curve modelling estimation [The cell cycle assay was conducted in accord with a previous study . Brieflytimation ,53.4 cells/mL) was seeded in a 100 mm culture dish for 24 h, followed by treatment with the IC25 and IC50 values of H2O2 and MIA, or IL-1β at 50 and 100 ng/mL for another 24 h. All the treated cells, including the floating cells, were then collected, trypsinised and washed twice with ice-cold PBS at 200 g for 5 min. The cells were then lysed with 200 µL of Qproteome mammalian lysis buffer  with protease inhibitors for 15 min on ice with gentle vortexing from time to time. The contents were then centrifuged at 4 °C and 10,000× g for 15 min. The supernatants were collected as lysates and kept at −80 °C until analysis. The protein concentrations of lysates were determined using Bio-Rad Protein Assay concentrated dye reagent  in microtiter format according to the manufacturer’s instructions. Bovine serum albumin (0–2 mg/mL) was used as the protein standard. The absorbance of each well was measured at 595 nm using a Multiskan GO microplate reader . All the protein concentrations of lysates (500 µL) were then diluted to 1 mg/mL for the subsequent quantitative detection of isoprostane and TNF-α levels.Briefly, 15.5 mL of SW1353 cells  and TNF-α  were determined using the sandwich-ELISA kits from Bioassay Technology Laboratory  and Elabscience . The procedures were conducted according to the manufacturer’s instructions ,55. For For human TNF-α quantification, 100 μL of lysates (1 mg/mL) was added in duplicate into a 96-well plate pre-coated with human TNF-α antibody. Similar amounts of standard solutions (0–500 pg/mL) were added separately into other wells. The plate was incubated at 37 °C for 90 min. All the content of wells was then removed and 100 μL of a biotinylated detection antibody working solution was added directly. The plate was further incubated at 37 °C for 1 h. After that, the plate was washed 3 times with a wash buffer and blot-dried with paper towels, then 100 μL of an HRP conjugate working solution was added. The plate was sealed and further incubated at 37 °C for another 30 min. After that, the plate was washed 5 times with a wash buffer and blot-dried with paper towels, then 90 μL of the substrate reagent was added. The plate was incubated at 37 °C for another 15 min. Lastly, 50 μL of the stop solution was added into each well and the absorbance of each well was measured at 450 nm by using a Multiskan GO microplate reader . The human TNF-α levels of lysates were determined based on the standard curve.p-value of less than 0.05 was considered statistically significant.The data were collected from three independent experiments with the respective technical replicates. Statistical analysis was conducted using SPSS software for Windows, version 25 . Normality tests confirmed the normal distribution of data. Mean differences among the study groups were analysed with one-way analysis of variance with Tukey’s or Dunnett’s test as post hoc analysis. A 2O2 on SW1353 chondrocytes upon 24 h of treatment. MIA (25 and 50 μM) and H2O2  were cytotoxic to SW1353 chondrocytes, with a concentration-dependent decline in cell viability . The IC25 and IC50 values for H2O2 treatment were 55 and 82.5 μM. The values were 22.5 and 31.5 μM for MIA treatment. IL-1β did not cause a significant decline in the viability of cells up to 100 ng/mL upon 24 h treatment (p > 0.05). Therefore, the highest and second-highest concentrations of IL-1β (50 and 100 ng/mL) were used in the subsequent experiment.2O2 and IL-1β treatment. SW1353 cells in the VC group demonstrated a typical spindle-shaped fibroblast-like morphology. Active and proliferating cells with a shiny appearance were also observed. Consistent with the cytotoxicity and mode of cell death findings, IL-1β did not cause any morphological changes in SW1353 chondrocytes compared with VC. Upon MIA treatment, there was no obvious change in SW1353 cell morphology, but the number of attached cells was reduced in a concentration-dependent manner. On the other hand, SW1353 chondrocytes treated with H2O2 demonstrated typical apoptotic features, including blebbing formation, cell shrinkage and apoptotic body formation. Besides, H2O2-treated SW1353 cells exhibited elongated or epithelial-shaped cells with reduced cell density.2O2 (both IC25 and IC50) significantly induced SW1353 cell death with a primarily increase in the apoptotic population (p < 0.001) and a slight increase in the necrotic population (p < 0.001). Parallel with the cytotoxicity data, IL-1β did not induce significant SW1353 cell death compared with the VC (p > 0.05). Surprisingly, MIA (IC25 and IC50) did not induce significant SW1353 cell death after 24 h of treatment (p > 0.05).Plasma membrane phosphatidylserine externalization is one of the earliest signs of apoptosis ,56. The 50) and serum-free media for 24 h. Serum deprivation is known to induce G1 arrest [p < 0.001), while the MIA treatment significantly increased the S-phase cell population (p < 0.05) with the lower G2-phase population, indicating a S-phase arrest.1 arrest . In this2O2 and IL-1β treatment. The 24 h treatment with MIA and H2O2 significantly reduced the TNF-α levels  compared with the VC, while the IL-1β treatment significantly increased the TNF-α levels  compared with the VC in a concentration-dependent manner. On the other hand, SW1353 chondrocytes treated with the IC25 and IC50 of H2O2 demonstrated significantly higher 8-isoprostane F2-α levels . A similar increasing trend was observed in MIA-treated SW1353 chondrocytes, but only the IC50 of MIA treatment showed statistical significance compared with the VC . Neither 50 nor 100 ng/mL IL-1β significantly altered the isoprostane levels in SW1353 chondrocytes (p > 0.05), despite a significant induction of TNF-α levels.8-isoprostane F2-α is one of the oxidative stress and lipid peroxidation markers, which is stably formed via non-enzymatic peroxidation of fatty acids, primarily arachidonic acid . Figure 2O2 induced concentration-dependent cytotoxicity and cell death, primarily via apoptosis in SW1353 chondrocytes, with the typical apoptotic features including blebbing formation, cell shrinkage and cell debris formation. Besides, there was a slight but significant increase (3–5%) in the necrotic cell population upon H2O2 treatment which might be due to the direct H2O2 damage to the cell membrane [2O2 also significantly increased the 8-isoprostane F2-α levels in SW1353 cells, which is coherent with its known oxidant activity [2O2 reduced the intracellular TNF-α levels in SW1353 chondrocytes. On the other hand, 24 h of MIA decreased the viability of SW1353 cells in a concentration-dependent manner but without significant cell death. MIA-treated SW1353 chondrocytes were morphologically similar to the VC but with fewer attached cells. Subsequently, we demonstrated that MIA treatment caused S-phase cell cycle arrest on SW1353 cells, with a higher level of 8-isoprostane F2-α. DNA damage and GSH depletion have been reported to cause S-phase arrest for cell repair process [In this study, 24 h of IL-1β was non-cytotoxic to SW1353 chondrocytes, without significant cell death up to 100 ng/mL. The cell morphology of IL-1β-treated SW1353 cells appeared similar to the control, consistent with the cytotoxicity and cell death findings. Besides, IL-1β-treated SW1353 cells were detected at a higher intracellular TNF-α level but insignificant changes to the 8-isoprostane F2-α (oxidative stress marker) level. On the other hand, 24 h of Hactivity ,37,38. H process . TherefoThe proinflammatory cytokine IL-1β was used to induce inflammation in SW1353 chondrocytes at the concentrations of 5–20 ng/mL, but its cytotoxic effect was not determined ,68,69,70There was a significant increase in intracellular TNF-α levels in IL-1β-treated cells. Mechanistically, IL-1β activates the activator protein-1 and nuclear factor kappa-light-chain-enhancer of activated B cell signalling pathway, subsequently upregulating the expression of downstream cytokines, including IL-1β and TNF-α . A 24 h Oxidative stress occurs when cell antioxidant defences are overwhelmed by ROS accumulation . Previou2O2 and MIA were cytotoxic and cytostatic to SW1353 chondrocytes, respectively. These changes were accompanied by an increase in 8-isoprostane F2-α levels but a lower level of intracellular TNF-α. Previous studies showed that the IC50 values of H2O2 in SW1353 chondrocytes ranged from 0.5 to 1 mM upon 1 h to 24 h of treatment [50 values around 5–8.26 µM for MIA were reported for SW1353 chondrocytes [50 values of H2O2 and MIA were 82.5 µM (6- to 12-fold lower) and 31.5 µM (4- to 6-fold higher), respectively. Multiple factors, such as batch-to-batch variation of cells and reagents, and culturing and treatment conditions, may have contributed to this discrepancy. The differences in media composition [50 value of MIA because it could provide an alternative energy source to the cells when glycolysis is inhibited [2O2 in our study might be due to the absence of pyruvate in the culture media, where 0.5–1 mM pyruvate  was shown to protect the cells from exogenous H2O2 [50 values of H2O2 from the media without pyruvate was about five-fold lower than in those containing pyruvate [Our study showed that Hreatment ,37,38,39drocytes ,43 and pdrocytes ,46,87. Oposition  may affenhibited . Additioous H2O2 ,91. Besi2O2 and MIA treatment significantly reduced the TNF-α levels in SW1353 chondrocytes despite higher 8-isoprostane F2-α levels. H2O2 [2O2- or MIA-treated SW1353 chondrocytes. The available studies showed that 24 h of H2O2 (0.5–10 mM) increased the release of TNF-α in SW1353 chondrocytes [2O2 treatment (40 ng/mL or 1.17 µM) also increased the intracellular TNF-α levels in primary rat articular chondrocytes [2O2 may interfere with the expression of TNF-α. Several proteinases, including caspases and calpains, are activated during apoptosis and they actively degrade the intracellular proteins and organelles [2O2 treatment in downregulating TNF-α levels.Both Hls. H2O2 ,37,38 anls. H2O2 ,46,87,92drocytes . A lowerdrocytes . MIA wasdrocytes ,92 and sganelles . FurtherThere are several limitations to this study. The use of single oxidative and inflammatory markers renders the interpretation of data non-comprehensive. The status of other cytokines or mediators like IL-6 and MMPs may be useful in validating the effects of OA inducers. Besides, direct measurement of ROS species is more informative than an indirect indication from the oxidative markers, although the ROS are extremely reactive and transiently present, which may be difficult to visualize . Time-co2O2 induced apoptosis while MIA induced a cytostatic effect on SW1353 cells, without a significant inflammation process. IL-1β is a suitable inducer of inflammation but not SW1353 cell death. Caution should be taken regarding the treatment conditions, especially the media composition and serum-free conditions in these in vitro OA models.In summary, our study demonstrated that H


Arabidopsis. The expression patterns and subcellular localization of the individual spliced variants of βCA1 were examined. The results showed that the spliced variants of βCA1 possessed different subcellular and tissue distributions and responded differently to environmental stimuli. Additionally, we addressed the physiological role of βCA1 in heat stress response and its protein-protein interaction (PPI) network. Our results showed that βCA1 was regulated by heat stresses, and βca1 mutant was hypersensitive to heat stress, indicating a role for βCA1 in heat stress response. Furthermore, PPI network analysis revealed that βCA1 interacts with multiple proteins involved in several processes, including photosynthesis, metabolism, and the stress response, and these will provide new avenues for future investigations of βCA1.Carbonic anhydrases (CAs) are ubiquitous zinc metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. Higher plants mainly contain the three evolutionarily distinct CA families αCA, βCA, and γCA, with each represented by multiple isoforms. Alternative splicing (AS) of the CA transcripts is common. However, there is little information on the spliced variants of individual CA isoforms. In this study, we focused on the characterization of spliced variants of βCA1 from  As CO2 is the main source of carbon, these CA enzymes are involved in crucial physiological processes, including almost all metabolic processes in higher plants and algae. Molecular, biochemical, and genetic studies of CAs in a wide range of tissues across diverse plant species have determined that CAs are involved in a wide range of diverse biological processes, including the provision of bicarbonate for anaplerotic reactions (Carbonic anhydrases (CAs) are a group of Zn-containing enzymes that catalyze the reversible hydration of carbon dioxide , and rice too has a similar number  , beta CAs (β), gamma CAs (γ) and delta CAs (δ) . Higher , 4 γCA) . However, 4 γCA) .Arabidopsis, including AtβCA1, AtβCA2, and AtβCA4, are predicted to encode at least two mRNA transcripts via AS  is an important regulatory mechanism that substantially expands eukaryotic transcriptome and proteome diversity and represents an additional level of cellular regulation . In manys via AS . Two splbidopsis . Furtherd leaves . AS of Ced by AS . AlthougβCA1 homologs between plant species, focusing on βCA1 spliced variants from Arabidopsis. We examined the expression patterns and subcellular localization of individual spliced variants of βCA1. We also analyzed the role of βCA1 in response to abiotic stress and the protein-protein interaction (PPI) network. This study will contribute to our understanding of βCA1.In this study, we investigated AS in Arabidopsis plants generated in this study are of the Columbia-0 (Col-0) ecotype. The T-DNA mutants lines used in this study have been described previously: βca1  and βca2 (CS303346) (All S303346) . Seeds wArabidopsis seedlings using ReliaPrep™ RNA Miniprep System . For cDNA synthesis, the first-strand cDNA was synthesized from 2 μg total RNA with NovoScript®Plus All-in-one 1st Strand cDNA Synthesis SuperMix  according to the instructions. For semi-quantitative RT-PCR analysis primers were designed manually after identification of unique sites in the cDNA of four spliced variants of βCA1 . Specific primers for amplification of βCA1.2 and βCA1.3 were applied, respectively. The primers used were mentioned in ACTIN2 was used as the endogenous control.Total RNA was extracted and purified from the 1-week-old Arabidopsis in the representative plant genome , the amino acid sequence of AtβCA1 was used as the guide sequence to perform a BLASTp search of the database whole genome sequences in the Phytozome database (https://phytozome.jgi.doe.gov). Coding sequences (CDS) and corresponding genomic DNA (gDNA) sequences of the βCA1s from the selected plant species were retrieved from the databases. The GSDS tool  was employed to analyze the exon-intron structures for plant βCA1 genes.To identify ortholog(s) of βCA1 from βCA1 spliced variants  were amplified and cloned into pDONOR221 using Gateway™ BP Clonase™ Enzyme Mix . Each βCA1 spliced variant was further cloned into the pGWB405 plant expression vector designed for the production of C-terminal GFP-tagged fusion proteins under the control of the 35S Cauliflower Mosaic Virus (35S CaMV) promoter using Gateway™ LR Clonase™ II Enzyme mix . To generate the plasmid for the native expression of βCA1, the genomic region including 1.5 kb upstream of ATG plus introns and exons were cloned. For insertion of specific tag including HA or Myc, overlapping PCR was applied by designed primers. These DNA fragments were cloned into pDONOR223 and pGWB4 vectors with the same method used for βCA1 spliced variants cloning. The constructs were confirmed by sequencing. All the plant expression constructs were introduced into the Agrobacterium tumefaciens GV3101 strain. Plant transformation was performed with floral dipping method . The CDS of four g method . All theTo analyze the GFP-tagged βCA1 spliced variants, seedlings of stable transgenic plants expressing the corresponding GFP fusions were freeze grounded into powder and homogenized in total protein buffer (20 mmo1/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 2 mmol/L EDTA) with protease inhibitor cocktail in DMSO . Lysates were incubated on ice for 20 min and clarified by centrifugation at 18,407 g for 15 min at 4 °C. For immunoblotting, samples were separated on 12% SDS polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked with 5% (g/v) defatted milk in TBST buffer (10 mM Tris-HCl (pH7.4), 150 mM NaCl, 0.05% Tween 20) and probed with using appropriate antibodies including 1:6,000 dilution α-GFP conjugated with HRP , 1:2,000 α-HA  and 1:2,000 α-Myc  overnight at 4 °C. Then the samples were washed with TBST buffer for three times and visualized by using the ECL . Actin protein was used as the internal control.The protoplasts from the 4-week-old stable transgenic plants leaves with GFP-tagged βCA1 were isolated following the previous used methods . The sub2, 20% glycerol, 0.2% CA-360, 1× protease inhibitor cocktail in DMSO). Lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4 °C and were incubated with Magnetic GFP beads  for overnight at 4°C in a top to end rotator. After incubation, the beads were washed five times with ice-cold washing buffer  and then eluted by boiling in reducing SDS sample buffer. Samples were separated by SDS-PAGE. For mass spectrometry analysis, the gel was stained with Coomassie blue after SDS-PAGE separation, and the visible stained protein bands were cut out for in-gel trypsin digestion, followed by tandem liquid chromatograph-mass spectrometry (LC-MS/MS) at Shanghai Applied Protein Technology Co. Ltd.The samples of 0.5 g 7-d-old seedlings tissue were freeze grounded to powder and homogenized in 2 ml IP buffer  under the following gene IDs: βCA1 (At3g01500), βCA1.1 (At3g01500.1), βCA1.2 (At3g01500.2), βCA1.3 (At3g01500.3), βCA1.4 (At3g01500.4).Sequence data from this article can be found in The Arabidopsis In-formation Resource , two dicots , and Physcomitrella patens. Further analysis of genome annotation obtained from the Phytozome database revealed that all βCA1 genes from selected plant species possessed several transcripts resulting from AS  had an HA tag (YPYDVPDYA) directly attached to the last exon before the stop codon. The second (βCA1:βCA1-Myc) had a Myc tag (QILFRDEFLL) linked to the end of the second-to-last exon, which was designed for expression analysis of four individual spliced variants of βCA1 in vivo , whereas salt and heat stresses significantly repressed the transcriptional expression of βCA1.2 and βCA1.3  and ABA on the expression pattern of d βCA1.3 . This inE. coli recombinants and that CA1 proteins from poplar were induced under heat stress  . These proteins played various roles, most of which were related to the functions of βCA1, including photosynthesis and stress response  . To gainresponse . Surprisresponse .Additional PPI networks for βCA1.4 associated proteins were constructed using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 11.0). The full set of 109 proteins produced an interactome map composed of 108 nodes (proteins) and 516 edges, with an average node degree of 9.56  . The varThe CA family plays various roles in many biological processes. Both mammalian and plant cells mainly possess two (α and β) or three  families with multiple isoforms. In each family, AS within individual isoforms further generates diversity, which significantly contributes to the structural and functional diversification of CA proteins. However, there is still a lack of information regarding AS of CA in plants.Arabidopsis was selected for a comprehensive study. We found that the βCA1 gene contained at least four spliced variants from all the representative plants, indicating a conserved evolutionary pattern for AS in βCA genes  seedlings might be due to posttranslational modification of βCA1 variants, as βCA1 has been reported to be phosphorylated by the leucine-rich repeat receptor-like kinase EXCESS MICROSPOROCYTES1 (EMS1) (βCA1:βCA1-HA (or βCA1:βCA1-Myc) seedlings in the absence or presence of alkaline phosphatase, suggesting two different protein sizes not result from phosphorylation modification (data not shown). On the other hand, different expression pattern of βCA1 variants were observed. The subcellular localization of them could be same if the mature βCA1.3 and βCA1.4 proteins are the same size. However, our results showed that localization of βCA1.3 is quite different from that of βCA1.4  . Howeverf βCA1.4 . In addiNmuCA1a from Neurachne are important for chloroplast targeting and have been suggested to be functional chloroplast transit peptides (Arabidopsis (KDVATILHWKL) was different from that in Neurachne (ASLGTPAPSSS) and was possibly involved in chloroplast transition. In addition, the short peptide from Arabidopsis is only found in CA genes from dicots, while the other one from Neurachne is known only from monocots, indicating evolutionary differences between these plants  and (2) this may bolster the potential physiological roles of CA.The correct inter- and intracellular placement of CAs is essential for efficient physiological function. A previous study showed that the long form of βCA4, βCA4.1, localizes to the plasma membrane while the short form, βCA4.2, is cytosolic . This supeptides . Howevere plants . This ree plants , 2B. In chondria . Moreove cytosol . As the Arabidopsis βCA1 has been reported to function together with βCA4 in disease resistance, and was induced under cold stress at the translational level . On the other hand, we found that loss-of-function βca1 mutants were much more sensitive to heat stress during seed germination . This indicates functional diversity in the CA family. Additionally, the contribution to heat stress tolerance of CA has been found in other plant species, such as rice and Pyropia haitanensis .AT3G01500 from Click here for additional data file.10.7717/peerj.12673/supp-2Supplemental Information 2A–B) dynamic GFP signals were observed from the mature zone of transgenic plants .(Click here for additional data file.10.7717/peerj.12673/supp-3Supplemental Information 3The protein accumulation of four βCA1 transcripts was analyzed by western blotting.Click here for additional data file.10.7717/peerj.12673/supp-4Supplemental Information 41-8: individual Arabidopsis seedlings, DL2000 was for the DNA ladder. Actin2 was used as the control.Click here for additional data file.10.7717/peerj.12673/supp-5Supplemental Information 5The proteins with localization predicted in stromule were in red.Click here for additional data file.10.7717/peerj.12673/supp-6Supplemental Information 6The specific short peptides required for chloroplast targeting were heighted in black boxes. D: dicot; M: monocot.Click here for additional data file.10.7717/peerj.12673/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.12673/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.12673/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.12673/supp-10Supplemental Information 10Click here for additional data file.