Journal: International Journal of Biological Sciences

Article Title: SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization

doi: 10.7150/ijbs.96563

Figure Lengend Snippet: SLC25A33 is upregulated in LPS/IFN-γ-induced M1 macrophages and CD14+ monocytes from septic patients. (A) Volcano plot illustrating gene expression differences between untreated PMs and LPS/IFN-γ-treated PMs. Red asterisks denote differentially expressed genes in the SLC25 family, and green asterisks highlight highly upregulated genes within the same family. (B-D) Relative mRNA expression of SLC25A33 , SLC25A22 , and SLC25A37 (B) and pro-inflammatory cytokines (C) in CD14+ monocytes from septic patients with liver abscesses (n=17) compared with healthy controls (n=10), and (D) changes in relative mRNA expression pre- and post-recovery in these septic patients. (E-F) Relative iNOS and SLC25A33 mRNA expression (E) and their protein levels (F) in LPS/IFN-γ-treated PMs. (G) An ex vivo experimental protocol for the isolation and harvesting of PMs from C57BL/6 mice treated with LPS. (H-J) Relative mRNA expression of iNOS and SLC25A33 (H), their corresponding protein expression levels (I), and relative mRNA expression of pro-inflammatory cytokines (J) in PMs isolated from mice following intraperitoneal LPS injection. In all in vitro experiments, PMs were treated with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 h. For ex vivo experiments, mice were injected intraperitoneally with LPS (2 mg/kg) and PMs were isolated after 24 h. All experimental data were verified in at least three independent experiments. Data are presented as the mean ± SEM. N.S., not significant, *p < 0.05, **p < 0.01 and ***p < 0.001. iNOS: inducible nitric oxide synthase; PMs: peritoneal macrophages.

Article Snippet: Cells were transfected with scramble siRNA, siSLC25A33, siMyD88, siPI3K, simTOR, siTAK1, siTBK1, siATF4 (Bioneer), SLC25A33 ORF Clone (Origene) or ATF4 ORF Clone (GeneCopoeia) using Lipofectamine TM 3000 (Thermo Fisher Scientific).

Techniques: Gene Expression, Expressing, Ex Vivo, Isolation, Injection, In Vitro

Journal: International Journal of Biological Sciences

Article Title: SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization

doi: 10.7150/ijbs.96563

Figure Lengend Snippet: SLC25A33 inhibition reduces LPS/IFN-γ-induced inflammatory response and mtROS of M1 macrophages. (A-B) Effect of SLC25A33 -targeting siRNA on iNOS mRNA expression (A) and protein levels of iNOS, CD80, and CD86 (B) in LPS/IFN-γ-treated PMs. (C-D) Effect of SLC25A33 -targeting siRNA on relative mRNA expression (C) and secretion (D) of pro-inflammatory cytokines in the LPS/IFN-γ-treated PMs. (E-F) Effect of PLP on iNOS mRNA expression (E) and protein levels of iNOS, CD80, and CD86 (F) in the LPS/IFN-γ-treated PMs. (G-H) Effect of PLP on the relative mRNA expression (G) and secretion (H) of pro-inflammatory cytokines in the PMs treated with LPS/IFN-γ. (I-J) Representative image of immunofluorescence staining for MitoSOX Red (left panel) and quantification of fluorescence intensity (right panel) in the LPS/IFN-γ-treated PMs following treatment with SLC25A33 -targeting siRNA (I) and PLP (J). In all experiments, PMs were treated with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 h, with PLP (400 μM, 24 h), MitoSOX Red (5 μM, 15 minutes), and SLC25A33 -targeting siRNA (40 pM; 48 h). All experimental data were verified in at least three independent experiments. Data are presented as the mean ± SEM. Scale bar represents 10 μm. *p < 0.05, **p < 0.01, and ***p < 0.001. iNOS: inducible nitric oxide synthase; PMs: peritoneal macrophages; PLP: pyridoxal 5'-phosphate.

Article Snippet: Cells were transfected with scramble siRNA, siSLC25A33, siMyD88, siPI3K, simTOR, siTAK1, siTBK1, siATF4 (Bioneer), SLC25A33 ORF Clone (Origene) or ATF4 ORF Clone (GeneCopoeia) using Lipofectamine TM 3000 (Thermo Fisher Scientific).

Techniques: Inhibition, Expressing, Immunofluorescence, Staining, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization

doi: 10.7150/ijbs.96563

Figure Lengend Snippet: MyD88-PI3K-mTORC1 signaling and ATF4 are essential drivers of SLC25A33 expression in M1 macrophages. (A) Relative mRNA expression of SLC25A33 in PMs treated with LPS, IFN-γ, or their combination. (B-D) Effect of the MyD88 inhibitor ST2825 and the TBK1 inhibitor MRT67307 on the levels of indicated proteins (B), as well as on relative mRNA expression (C) and protein levels (D) of SLC25A33 in the LPS/IFN-γ-treated PMs. (E-G) Effect of the PI3K inhibitor LY294002 or the TAK1 inhibitor Takinib on the levels of the indicated proteins (E) and their relative mRNA expression (F) and the protein levels (G, LY294002 only) of SLC25A33 in the LPS/IFN-γ-treated PMs. (H) Effect of rapamycin on the relative mRNA expression of SLC25A33 in the LPS/IFN-γ-treated PMs. (I) Effect of rapamycin or LY294002 on protein levels of phosphorylated-S6K, S6K, SLC25A33, and ATF4 in the LPS/IFN-γ -treated PMs. (J) Effect of MyD88 , PI3K , mTOR , TAK1 or TBK1 -targeting siRNAs on protein levels of ATF4 and SLC25A33 in the LPS/IFN-γ-treated PMs. (K-M) Effect of ATF4 -targeting siRNA on the relative mRNA expression of ATF4 (K), SLC25A33 (L) and protein levels of ATF4 and SLC25A33 in the LPS/IFN-γ-treated PMs (M). (N) Effect of ATF4 -targeting siRNA on the relative luciferase activity of the Gluc reporter in the LPS/IFN-γ-treated PMs. (O) ChIP assay of ATF4 binding to its putative site within the SLC25A33 promoter in LPS/IFN-γ-treated PMs. (P) Effect of mutation of the putative ATF4 binding site within the SLC25A33 promoter on the relative luciferase activity of the Gluc reporter in the LPS/IFN-γ-treated PMs. In all experiments, PMs were treated with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 h, with ST2825 (10 μM, 24 h), MRT67307 (1 μM, 24 h), LY294002 (25 nM, 24 h), Takinib (10 μM, 24 h), rapamycin (50 nM, 24 h), and siRNAs against each gene (40 pM, 48 h). All experimental data were verified in at least three independent experiments. Data are presented as the mean ± SEM. N.S., not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. ATF4: activating transcription factor 4; Gluc: Gaussia luciferase; PMs: peritoneal macrophages.

Article Snippet: Cells were transfected with scramble siRNA, siSLC25A33, siMyD88, siPI3K, simTOR, siTAK1, siTBK1, siATF4 (Bioneer), SLC25A33 ORF Clone (Origene) or ATF4 ORF Clone (GeneCopoeia) using Lipofectamine TM 3000 (Thermo Fisher Scientific).

Techniques: Expressing, Luciferase, Activity Assay, Binding Assay, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization

doi: 10.7150/ijbs.96563

Figure Lengend Snippet: SLC25A33 inhibition reduces mitochondrial DNA release and modulates the cGAS-STING pathway in M1 macrophages. (A-B) Effects of SLC25A33 -targeting siRNA (A) and PLP (B) on relative mtDNA levels in LPS/IFN-γ-treated PMs. (C-D) Representative immunofluorescence images using 6- O -Propynyl-dG (PdG) and click chemistry (left panel) and quantification of PdG (right panel) in the LPS/IFN-γ-treated PMs following treatment with SLC25A33 -targeting siRNA (C) and PLP (D). (E) Representative immunofluorescence images of dsDNA and mitochondria (left panel) and quantification of cytosolic dsDNA foci (right panel) in the LPS/IFN-γ-treated PMs with SLC25A33 -targeting siRNA. (F) Effect of SLC25A33 -targeting siRNA on relative cytosolic mtDNA levels in LPS/IFN-γ-treated PMs, measured by qPCR. (G) Representative immunofluorescence images of dsDNA and mitochondria (left panel) and quantification of cytosolic dsDNA foci (right panel) in the LPS/IFN-γ-treated PMs with PLP. (H) Effect of PLP on the relative cytosolic mtDNA levels in LPS/IFN-γ-treated PMs, measured by qPCR. (I-J) Effects of SLC25A33 -targeting siRNA (I) and PLP (J) on phosphorylation status of STING, p65, and IRF3 in the LPS/IFN-γ-treated PMs. (K) Effect of ATF4 overexpression on protein levels of SLC25A33 and phosphorylation status of IKKα/β, IκBα, and p65 in SLC25A33 knock-downed LPS/IFN-γ-treated PMs. (L) Effect of SLC25A33 overexpression on phosphorylation of cGAS-STING pathway proteins in ATF4 knock-downed LPS/IFN-γ-treated PMs. In all experiments, PMs were treated with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 h, with PLP (400 μM, 24 h), MitoTracker (250 nM, 30 minutes), PdG (10 μM, 1 h), and siRNAs against each gene (40 pM, 48 h). All experimental data were verified in at least three independent experiments. Data are presented as the mean ± SEM. Scale bar represents 10 μm. *p < 0.05, **p < 0.01, and ***p < 0.001. dsDNA: double-stranded DNA; PLP: pyridoxal 5'-phosphate; IRF3: interferon resgulatory factor 3; STING: stimulator of interferon genes.

Article Snippet: Cells were transfected with scramble siRNA, siSLC25A33, siMyD88, siPI3K, simTOR, siTAK1, siTBK1, siATF4 (Bioneer), SLC25A33 ORF Clone (Origene) or ATF4 ORF Clone (GeneCopoeia) using Lipofectamine TM 3000 (Thermo Fisher Scientific).

Techniques: Inhibition, Immunofluorescence, Phospho-proteomics, Over Expression

Journal: International Journal of Biological Sciences

Article Title: SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization

doi: 10.7150/ijbs.96563

Figure Lengend Snippet: SLC25A33 increases mtDNA release via VDAC1 oligomerization and mtROS in M1 macrophages. (A-C) Effect of ddC (A), SLC25A33 -targeting siRNA (B), and PLP (C) on VDAC1 oligomerization, as indicated by immunoblotting positions for monomers (Mono), trimers (Tri), tetramers (Tetra), and multimers (Multi). Asterisk indicates a nonspecific band. (D) Representative image of immunofluorescence staining for dsDNA and mitochondria (left panel) and quantification of cytosolic dsDNA foci (right panel) in LPS/IFN-γ-treated PMs with VBIT-4. (E) Effect of VBIT-4 on the protein expression level of iNOS, CD80, and CD86 in the LPS/IFNγ-treated PMs. (F) Effect of VBIT-4 on the mRNA expression of iNOS and pro-inflammatory cytokines in the LPS/IFNγ-treated PMs. (G) Representative image of immunofluorescence staining for MitoSOX Red in the LPS/IFN-γ-treated PMs with VBIT4. (H) Effect of MitoTEMPO on VDAC1 oligomerization. Asterisk indicates a nonspecific band. (I) Representative image of immunofluorescence staining for dsDNA and mitochondria (left panel) and quantification of cytosolic dsDNA foci (right panel) in the LPS/IFN-γ-treated PMs with MitoTEMPO. In all experiments, PMs were treated with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 h, with ddC (40 μM, 5 days), PLP (400 μM, 24 h), VBIT-4 (10 μM, 48 h), MitoTEMPO (500 μM, 24 h), MitoSOX Red (5 μM, 15 minutes), and SLC25A33 -targeting siRNAs (40 pM, 48 h). All experimental data were verified in at least three independent experiments. Data are presented as the mean ± SEM. Scale bar represents 10 μm. *p < 0.05, **p < 0.01, and ***p < 0.001. ddC: 2′,3′-dideoxycytidine; dsDNA: double-stranded DNA; EGS: ethylene glycol-bis(succinimidyl succinate); VDAC1: voltage-dependent anion channel 1; PLP: pyridoxal 5'-phosphate.

Article Snippet: Cells were transfected with scramble siRNA, siSLC25A33, siMyD88, siPI3K, simTOR, siTAK1, siTBK1, siATF4 (Bioneer), SLC25A33 ORF Clone (Origene) or ATF4 ORF Clone (GeneCopoeia) using Lipofectamine TM 3000 (Thermo Fisher Scientific).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization

doi: 10.7150/ijbs.96563

Figure Lengend Snippet: Reconstitution with SLC25A33 -silenced BMDMs mitigates inflammation in clodronate liposome (CL) treated mice during LPS-induced sepsis. (A) Schematic representation of the procedure for the isolation of BMDMs from C57BL/6 male mice, transduction of SLC25A33 -targeting shRNA, and their reconstitution into macrophage-depleted mice treated with 200 μL CL. (B) SLC25A33 protein levels in SLC25A33 -silenced BMDMs before their introduction into CL-treated mice. (C) Quantification of macrophages in mice 2 days post CL treatment. (D-E) Effect of SLC25A33 -silenced BMDMs reconstitution on the survival rate (n=8) (D) and pro-inflammatory cytokines (E) in LPS (30 mg/kg) induced septic mice. (F) Representative images of H&E staining (upper panel) (Scale bar represents 100 μm), immunohistochemical staining with anti-CD86 antibodies (lower panel) (Scale bar represents 20 μm), and the CD86 expression scores (right panel) determined using ImageJ in lung tissue sections from LPS-induced septic mice reconstituted with or without the SLC25A33 -silenced BMDMs. All experimental data were verified in at least three independent experiments for each animal group. Data represent the mean ± SEM from independent mice. *p < 0.05, **p < 0.01, and ***p < 0.001. BMDMs: bone marrow-derived macrophages.

Article Snippet: Cells were transfected with scramble siRNA, siSLC25A33, siMyD88, siPI3K, simTOR, siTAK1, siTBK1, siATF4 (Bioneer), SLC25A33 ORF Clone (Origene) or ATF4 ORF Clone (GeneCopoeia) using Lipofectamine TM 3000 (Thermo Fisher Scientific).

Techniques: Isolation, Transduction, shRNA, Staining, Immunohistochemical staining, Expressing, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: SLC25A33-mediated mitochondrial DNA synthesis plays a critical role in the inflammatory response of M1 macrophages by contributing to mitochondrial ROS and VDAC oligomerization

doi: 10.7150/ijbs.96563

Figure Lengend Snippet: PLP alleviates systemic inflammatory responses in LPS- and CLP-induced septic mice. (A) Effect of PLP (20 mg/kg) on the survival rate of septic mice induced by LPS (30 mg/kg, n=6). (B-C) Relative mRNA levels of pro-inflammatory cytokines in PMs isolated LPS-induced (B) and their relative cytokines levels in the serum of LPS-induced (C) septic mice treated with PLP. (D) Representative images of H&E staining (upper panel) and immunohistochemical staining (lower panel) with antibodies against CD86 in lung tissue in the LPS-induced septic mice (Scale bar represents 100 μm). (E) Representative immunofluorescence images for CD80 (red) and SLC25A33 (green) in lung tissue from the LPS-induced septic mice treated with PLP (Scale bar represents 30 μm). (F) Effect of PLP on the survival rate of septic mice induced by CLP (n=7). (G-H) Relative mRNA levels of pro-inflammatory cytokines in the colon of CLP-induced (G) and their relative protein levels in the serum of CLP-induced (H) septic mice treated with PLP. (I) Representative images of H&E staining (upper panel) (Scale bar represents 100 μm) and immunohistochemical staining (lower panel) (Scale bar represents 40 μm) with antibodies against CD86 in colon tissue in CLP-induced septic mice. (J) Representative immunofluorescence images for CD80 (red) and SLC25A33 (green) in colon tissue from CLP-induced septic mice treated with PLP (Scale bar represents 40 μm). All experimental data were verified in at least three independent experiments for each animal group. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. CLP: cecal ligation and puncture; PLP: pyridoxal 5'-phosphate.

Article Snippet: Cells were transfected with scramble siRNA, siSLC25A33, siMyD88, siPI3K, simTOR, siTAK1, siTBK1, siATF4 (Bioneer), SLC25A33 ORF Clone (Origene) or ATF4 ORF Clone (GeneCopoeia) using Lipofectamine TM 3000 (Thermo Fisher Scientific).

Techniques: Isolation, Staining, Immunohistochemical staining, Immunofluorescence, Ligation

Journal: Stem Cells International

Article Title: Vascularization of a Bone Organoid Using Dental Pulp Stem Cells

doi: 10.1155/2023/5367887

Figure Lengend Snippet: Comparison of the sprouting ability of BMSCs and DPSCs. (a) Photomicrographs of sprouting capillary structures formed by BMSCs and DPSCs with endothelial induction. (b) Quantitative analyses of the number of branches and (c) total branching length at corresponding time points. Scale bars: 200 μ m. ∗ p < 0.05; mean ± SD; n = 4.

Article Snippet: Human BMSCs derived from iliac crests (Lonza, Basel, Switzerland) and DPSCs isolated from adult third molars (Lonza) were cultured in growth medium (GM) comprising Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan) supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, MO, USA).

Techniques: Comparison

Journal: Stem Cells International

Article Title: Vascularization of a Bone Organoid Using Dental Pulp Stem Cells

doi: 10.1155/2023/5367887

Figure Lengend Snippet: Comparison of endothelial differentiation ability of BMSCs and DPSCs. mRNA expression of (a) VEGFA , (b) CXCL1 , and (c) Nanog after 1, 3, 7, and 14 days of endothelial induction. Different capital letters indicate significant differences among groups ( p < 0.05); mean ± SD; n = 4.

Article Snippet: Human BMSCs derived from iliac crests (Lonza, Basel, Switzerland) and DPSCs isolated from adult third molars (Lonza) were cultured in growth medium (GM) comprising Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan) supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, MO, USA).

Techniques: Comparison, Expressing

Journal: Stem Cells International

Article Title: Vascularization of a Bone Organoid Using Dental Pulp Stem Cells

doi: 10.1155/2023/5367887

Figure Lengend Snippet: Structure of the cell constructs after endothelial and osteogenic induction. (a) HE staining, (b) von Kossa staining, and (c) fluorescence imaging of cell constructs with varying ratios of BMSCs and DPSCs. White arrows indicate a capillary-like hollow structure in the mineralized 80B/20D constructs. Dotted lines indicate the outline of the cell constructs. Scale bars: 50 μ m.

Article Snippet: Human BMSCs derived from iliac crests (Lonza, Basel, Switzerland) and DPSCs isolated from adult third molars (Lonza) were cultured in growth medium (GM) comprising Dulbecco's modified Eagle's medium (DMEM; Wako, Osaka, Japan) supplemented with 20% fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, MO, USA).

Techniques: Construct, Staining, Fluorescence, Imaging

Journal: Stem Cell Research & Therapy

Article Title: Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation

doi: 10.1186/s13287-024-03803-3

Figure Lengend Snippet: Impact of Denosumab treatment on the mineralization capacity of human BMSCs. a Representative image of ARS-stained cultures of human BMSCs from healthy donors after 2 weeks of culture either in basal medium (CTR) or in OIM alone or in OIM + isotype control (OIM + ISO) or in OIM + Denosumab (OIM + Dmab); in all the conditions, the culture medium was changed twice a week. At the end, the mineral deposition was quantified and indicated by Abs reading at 405 nm. Scale bar: 500 μm. Higher magnifications are provided in Figure . b Gene expression analysis of representative osteogenic genes in human BMSCs in the different treatment conditions indicated; normalization on 18 S. Results are expressed as Arbitrary Units (A.U.). All the data are represented as mean ± SEM. * p < 0.05; a: Friedman test; b: Kruskal-Wallis test. CTR: control. OIM: Osteogenic Induction Medium. ISO: isotype control. Dmab: Denosumab

Article Snippet: Human BMSCs from young healthy individuals of both sexes were purchased from Stem Cell Technologies (cat. #70,071).

Techniques: Staining, Control, Expressing