Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: HMOX-1-specific targeting in KCs induces ferroptosis. (A) Schematic diagram illustrating the influence of HMOX-1 through ZnPP, CoPP, and CoPP + DFO. (B) Survival curves of mice pretreated with 10 mg/kg ZnPP, 5 mg/kg CoPP, or 5 mg/kg CoPP + 100 mg/kg DFO followed by HS ( n = 15 mice per group). Evaluation of hepatocellular function by AST (C), ALT (D), and TBIL (E) ( n = 3–5 mice per group). (F) Representative H&E staining of liver paraffin sections from HS mice (scale bar: 200 μm). Detection of MDA (G), tissue non-heme iron (H) ( n = 3–5 mice per group), and BODIPY 581/591 C11 (I) in liver tissue of HS mice (scale bar: 200 μm). (J) Schematic diagram illustrating the influence of HMOX-1 through specific Hmox1 knockout in KCs. (K) Survival curves of Clec4f-crexHmox1 flox/flox mice or Hmox1 flox/flox mice, followed by HS ( n = 15 mice per group). Measurement of ALT (L), AST (M), and TBIL (N) ( n = 3–5 mice per group). (O) Representative H&E staining of liver paraffin sections of Clec4f-cre x Hmox1 flox/flox mice or Hmox1 flox/flox mice (scale bar: 200 μm). Measurement of MDA (P), tissue non-heme iron (Q) ( n = 3–5 mice per group), and BODIPY 581/591 C11 (R) in liver tissue (scale bar: 200 μm). Summary data are presented as the mean ± SEM. Significance was calculated using a one-way ANOVA with Tukey's post hoc test. Significance in (B) and (K) is determined by the log-rank (Mantel–Cox) test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Staining, Knock-Out

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: HMOX-1 affects NLRP3 inflammasome activation. (A) Representative immunofluorescence staining images displaying the expression of Clec4F (purple), HMOX-1 (green), NLRP3 (red), and in the liver of mice after HS, with magnified insets (scale bar: 10 μm). (B) Western Blotting analysis of Caspase-1 in liver tissues of HS mice and statistical results ( n = 3) (C). (D) Representative images of immunofluorescence staining for Clec4F (purple), HMOX-1 (green), and NLRP3 (red) in liver tissues of HS mice pretreated with PBS, 10 mg/kg ZnPP, 5 mg/kg CoPP, or 5 mg/kg CoPP + 100 mg/kg DFO, with magnified insets (scale bar: 10 μm). (E) estern blotting analysis of caspase-1 in liver tissues of HS mice and statistical results ( n = 3) (F). (G) Measurement of plasma IL-1 β content by ELISA ( n = 3–5 mice per group). (H) Representative images of immunofluorescence staining for Clec4F (purple), HMOX-1 (green), and NLRP3 (red) in liver tissues of Clec4f-cre x Hmox1 flox/flox mice or Hmox1 flox/flox mice, with magnified insets (scale bar: 10 μm). (I) Western blotting analysis of caspase-1 in liver tissues and statistical results ( n = 3) (J). (K) Measurement of plasma IL-1 β content by ELISA ( n = 3–5 mice per group). Significance was calculated using a one-way ANOVA with Tukey's post hoc test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: HMOX-1 affects PI4K β activation in vitro . (A) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), TGN38 (red), and DAPI (blue) in KC2 treated at 43 °C for 3 h and recovered at 37 °C for 0, 6, or 24 h (scale bar: 5 μm), and statistical analysis (B) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (C) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), TGN38 (red), and DAPI (blue) in KC2 pretreated with 4 μmol/L ZnPP for 12 h or 4 μmol/L CoPP for 12 h (scale bar: 5 μm), and statistical analysis (D) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (E) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), PI4K β (red), and DAPI (blue) in KC2 (scale bar: 5 μm), and statistical analysis (F) of co-localization of OSBP-PH-GFP and PI4K β ( n = 20). (G) Representative images of immunofluorescence staining for OSBP-PH-GFP (green), TGN38 (red), and DAPI (blue) in KC2 pretreated with 5 μmol/L PI4K β inhibitor for 1 h (scale bar: 5 μm), and statistical analysis (H) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (I) Representative images of immunofluorescence staining for NLRP3 (red), TGN38 (green), and DAPI (blue) in KC2 (scale bar: 5 μm), and statistical analysis (J) of co-localization of OSBP-PH-GFP and TGN38 ( n = 20). (K) Representative images of immunofluorescence staining for PI4K β (red), TGN38 (green), and DAPI (blue) in KC2 pretreated with 4 μmol/L ZnPP for 12 h, 4 μmol/L CoPP for 12 h or 4 μmol/L CoPP for 12 h + 1 μmol/L Fer-1 for 1 h (scale bar: 5 μm), and statistical analysis (L) of co-localization of PI4K β and TGN38 ( n = 20). (M) Western blotting analysis of caspase-1 in KC2 pretreated with DMSO or PI4K β inhibitor and (N) statistical results ( n = 3). (O) Representative images of immunofluorescence staining for PI4K β (red), TGN38 (green), and DAPI (blue) in vector , sh-Hmox1 or OE-Hmox1 treated with 43 °C for 3 h and recovered at 37 °C for 6 h (scale bar: 5 μm) and (P) statistical analysis of co-localization of PI4K β and TGN38 ( n = 20). Summary data are presented as the mean ± SEM. Significance was calculated using a one-way ANOVA with Tukey's post hoc test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Activation Assay, In Vitro, Immunofluorescence, Staining, Western Blot, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

doi: 10.1016/j.apsb.2024.05.007

Figure Lengend Snippet: EGR1 regulates the transcription of Hmox1 . (A) Representative images of immunofluorescence staining for HMOX-1 (red), EGR1 (green), and DAPI (blue) in KC2 treated at 43 °C for 3 h and recovered at 37 °C for 0, 6, or 24 h (scale bar: 5 μm). (B) Western blotting analysis of the expression of HMOX-1 after knocking down Egr1 ( sh-Egr1 ) in ImKCs and statistical analysis ( n = 3) (C). (D) Representative images of immunofluorescence staining for PI4K β (red), TGN38 (green), and DAPI (blue) in vector or sh-Egr1 (scale bar: 5 μm). (E) Representative images of immunofluorescence staining for NLRP3 (red), TGN38 (green) and DAPI (blue) (scale bar: 5 μm). (F) Western blotting analysis of caspase-1. (G) Cellular supernatant IL-1 β content detected by ELISA ( n = 3 mice per group). (H) Dual-luciferase experiments in HEK-293T cells transfected with different truncated regions of the Hmox1 promoter ( n = 3). (I) Dual-luciferase experiments in HEK-293T cells transfected with different mutational regions of the Hmox1 promoter ( n = 3). ChIP-qRT-PCR analysis of EGR1 binding to −103 to −93, −1300 to −1290, −1849 to −1839 sites in the Hmox1 promoter region ( n = 3) (J) and fold change of signals in ImKC treated with 43 °C for 3 h and recovered at 37 °C for 6 h ( n = 3) (K). (L) A hypothetical model for KC2 ferroptosis in HS mice. Summary data are presented as the mean ± SEM. Significance was calculated using a one-way ANOVA with Tukey's post hoc test.

Article Snippet: Genetic Modifications: Mice with specific knockout of Hmox1 in KCs were generated by crossing C57BL/6J- Clec4f em1(cre)Glass /J (033296, Jackson lab) with C57BL/6J- Hmox1 em1Cflox /Cya (S–CKO-02911, Cyagen Biosciences) mice, resulting in Clec4f-cre x Hmox1 flox/flox mice.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Transfection, Quantitative RT-PCR, Binding Assay

Journal: Biomedicines

Article Title: Heme Oxygenase-1 Inhibition Modulates Autophagy and Augments Arsenic Trioxide Cytotoxicity in Pancreatic Cancer Cells.

doi: 10.3390/biomedicines11092580

Figure Lengend Snippet: Figure 1. Induction of HO-1 in human PDAC cells upon treatment with ATO. (A) The effect of ATO on HO-1 expression was examined through Western blot analysis in PDAC cells MiaPaca-1 and Capan-1. Cells were treated with different concentrations of ATO for 24 h. Protein was collected and run for immunoblotting as described in the Section 2. Densitometric analysis is shown next to each blot (n = 3, p < 0.05). (B) Increased HO-1 expression after ATO treatment in Colo-357 shown by immunoblotting. Confocal microscopy confirms ATO induction of HO-1 in Colo-357. (C) HO-1 upregulation is via activation of the p38 MAPK pathway in response to ATO. MiaPaca-2 and Capan-1 cells were treated with indicated concentrations of ATO to follow pp38 MAPK and HO-1 expression using Western blot analysis. Data are representative of three independent experiments. (** p < 0.01, **** p < 0.0001. n = 3).

Article Snippet: PDAC cells were transfected with an HMOX1 Human shRNA Plasmid Kit (Origene, MD, USA) and Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Confocal Microscopy, Activation Assay

Journal: Biomedicines

Article Title: Heme Oxygenase-1 Inhibition Modulates Autophagy and Augments Arsenic Trioxide Cytotoxicity in Pancreatic Cancer Cells.

doi: 10.3390/biomedicines11092580

Figure Lengend Snippet: Figure 2. Time- and dose-dependent ATO effects on PDAC cells’ survival. Cell survival and growth inhibition was measured using the MTT assay. (A) MiaPaca-2 pancreatic cell line was treated with indicated concentration of ATO (5–15 µM) for 24 h. (B) MiaPaca-2 cells’ survival after 24 and 48 h culture in combination with HO-1 inhibition. (C) Capan-1 response to ATO after 24 h incubation and (D) combining ATO with HO-1 inhibition for 24 and 48 h. (E) T3M4 PDAC cells’ response to ATO and HO-1 inhibition after 24 h. (F) Survival of parent and HO-1 KO MaiaPaca-2 cells in response to ATO as compared to own controls. Insert showing HO-1 KO in response to CoPP for 24 h as compared to parent cells. (** p < 0.01, **** p < 0.0001. n = 3).

Article Snippet: PDAC cells were transfected with an HMOX1 Human shRNA Plasmid Kit (Origene, MD, USA) and Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA).

Techniques: Inhibition, MTT Assay, Concentration Assay, Incubation

Journal: Biomedicines

Article Title: Heme Oxygenase-1 Inhibition Modulates Autophagy and Augments Arsenic Trioxide Cytotoxicity in Pancreatic Cancer Cells.

doi: 10.3390/biomedicines11092580

Figure Lengend Snippet: Figure 3. ATO and HO-1 inhibition promoted apoptosis in PDAC cells. PDAC cells were treated with vehicle, HO-1 inhibitor, and/or ATO. Apoptotic cells were counted using flow cytometry. (A) Example of flow results for T3M4 PDAC cells treated with either ATO, HO-1 inhibitor (SnPP), or combination. (B) Densitometric analysis paragraphs of total apoptotic cells of T3M4. (C) Apoptosis in T3M4 cells treated with the HO-1 inhibitor (ZnPP) and ATO. (D) Statistical paragraphs of T3M4 apoptosis in (C). (E) Apoptosis in MiaPaca-2 with the HO-1 inhibitor ZnPP and ATO. (F) Statistical paragraphs of MiaPaca-2 apoptosis in (E) (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).

Article Snippet: PDAC cells were transfected with an HMOX1 Human shRNA Plasmid Kit (Origene, MD, USA) and Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA).

Techniques: Inhibition, Cytometry

Journal: Biomedicines

Article Title: Heme Oxygenase-1 Inhibition Modulates Autophagy and Augments Arsenic Trioxide Cytotoxicity in Pancreatic Cancer Cells.

doi: 10.3390/biomedicines11092580

Figure Lengend Snippet: Figure 4. Effects of ATO, HO-1 inhibition, and combination on the levels of intracellular ROS as measured through confocal microscopy using an oxidation-sensitive fluorescent probe, 2′,7′- dichlorodihydrofluorescein diacetate (DCFH-DA), and Dihydroethidium (DHE). (A) ATO increased DCF fluorescence in MaiPaca-2 cells when treated as described in the Section 2. (B) Densitometric analysis of results in (A). (C) Increased DHE fluorescence in MiaPaca-2 cells after ATO treatment. (D) Oxidized glutathione (GSSG) in MiaPaca-2 and Capan-1 cells treated with ATO, HO-1 inhibitor, or combination. (E) Effects of NAC on ATO-induced antiproliferative effects in Colo-357, CD18/HPAF, and MiaPac-2 cells (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3). Scale bar: 20 µm.

Article Snippet: PDAC cells were transfected with an HMOX1 Human shRNA Plasmid Kit (Origene, MD, USA) and Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA).

Techniques: Inhibition, Confocal Microscopy

Journal: Biomedicines

Article Title: Heme Oxygenase-1 Inhibition Modulates Autophagy and Augments Arsenic Trioxide Cytotoxicity in Pancreatic Cancer Cells.

doi: 10.3390/biomedicines11092580

Figure Lengend Snippet: Figure 5. ATO and HO-1 inhibition disrupts lysosomal accumulation. Representative fluorescence images of LysoTracker Red staining of MiaPaca-2 cells for control and ATO- and CQN-treated cells (A) Both ATO and inhibiting HO-1 increased the fluorescence intensity of LysoTracker Red. (B) LysoTracker densitometry results show significantly increased lysosomal levels for cells treated with HO-1 inhibitors or CQN in comparison to the untreated control (** p < 0.01, *** p < 0.001, n = 3). Scale bar: 20 µm.

Article Snippet: PDAC cells were transfected with an HMOX1 Human shRNA Plasmid Kit (Origene, MD, USA) and Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA).

Techniques: Inhibition, Staining, Control, Comparison

Journal: Biomedicines

Article Title: Heme Oxygenase-1 Inhibition Modulates Autophagy and Augments Arsenic Trioxide Cytotoxicity in Pancreatic Cancer Cells.

doi: 10.3390/biomedicines11092580

Figure Lengend Snippet: Figure 6. Inhibiting HO-1 suppresses autophagy. (A) Fluorescence photographs of MiaPaca-2 cells treated with ATO (5 µM), SnPP (10 µM), NAC (1 mM), chloroquine (15 µM), or combinations for 24 h. Nuclei were stained with DAPI (blue). Cells were treated with DMSO as a control. ATG5 was used as autophagy marker. (B) Average fluorescence of autophagosomes (ATG5 positive) per cell was calculated. Scale bar: 20 µm. (C) Immunoblotting of autophagy proteins for cells treated with ATO and/or HO-1 inhibitors showing increased LC3, ATg5, and p62 protein expression in response to ATO and HO-1 inhibition as compared to control. Densitometric analysis of each protein is shown as compared to control (* p < 0.05, ** p < 0.01, **** p < 0.0001, n = 3). Scale bar: 20 µm.

Article Snippet: PDAC cells were transfected with an HMOX1 Human shRNA Plasmid Kit (Origene, MD, USA) and Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA).

Techniques: Fluorescence, Staining, Control, Marker, Western Blot, Expressing, Inhibition