Journal: Journal of Clinical Investigation

Article Title: Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells

doi: 10.1172/jci44855

Figure Lengend Snippet: Figure 1 “Mitochondriotoxic” activity of G-TPP. (A) Cultured glioblastoma cell lines (U87, purple; LN229, blue; U251, black), patient-derived glioblastoma cells (GS620, yellow; GS48, green; AS515, red), or naive (gray), or SV40-transformed (light blue) normal FHAS were incubated with GamitrinibTPP (G-TPP, top) or 17-AAG (bottom) and analyzed for cell viability by MTT. Mean ± SD of replicates. (B) U87 cells were treated with G-TPP or 17-AAG and analyzed for mitochondrial membrane potential by JC-1 staining and flow cytometry. FL1, green fluorescence channel; FL2, red fluorescence channel. (C and D) U87 cells were incubated with 0–20 μM G-TPP (C) or 20 μM G-TPP or 17-AAG (D) and analyzed by immunoblotting. Cyto c, cytochrome c; Casp., caspase; Cl., cleaved. COX-IV was used as a mitochondrial marker. (E) U251 cells were treated as indicated and analyzed for annexin V/PI staining by flow cytometry. None, untreated. For B and E, the percentage of cells in each quadrant is indicated.

Article Snippet: Antibodies to LC3 (Cell Signaling), CypD, COX-IV (1:1000; Calbiochem), cytochrome c (1:1000; Clontech), Hsp70 (1:1000; Abcam), TRAP-1 (BD), Ki-67 (Zymed; Invitrogen), caspase-9 (1:250; CST), caspase-8 (1:250; CST), caspase-3 (1:100; CST), caspase-7 (1:500; CST), FLIP-L, long form (1:250; CST) FLIP-S, short form (1:250; CST), CrmA (SCBT), β-actin (1:2000; Sigma-Aldrich), mitochondrial superoxide dismutase-2 (SOD2; 5 μg/ml; Sigma-Aldrich), survivin (NOVUS Biologicals), Bcl-2 (1:1000; CST), Bax (1:1000; CST), PUMA (1:1000; CST), Bcl-XL (1:1000; CST), Bak (1:1000; CST), Bad (1:1000; CST), Bid (1:1000; CST), Mcl-1 (1:1000; CST), C/EBP/β (1:1000; CST), CHOP (1:1000; CST), TAK1 (1:1000; Santa Cruz Biotechnology Inc.), α-glial fibrillary acidic protein (Dako), CD31 (BD Biosciences — Pharmingen) or factor VIII (Dako), or neuronal neurofilament proteins 70, 160, and 200 (all from Progen) were used.

Techniques: Activity Assay, Cell Culture, Derivative Assay, Transformation Assay, Incubation, Membrane, Staining, Flow Cytometry, Fluorescence, Western Blot, Marker

Journal: Journal of Clinical Investigation

Article Title: Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells

doi: 10.1172/jci44855

Figure Lengend Snippet: Figure 3 Mitochondrial Hsp90 regulates autophagy. (A–C) LN229 cells were treated with G-TPP and analyzed by EM (A and B) or immunoelectron microscopy with an antibody to COX-IV (C). Arrowheads, COX-IV–reactive material. Scale bars: 2 μm (A); 1 μm (B); 200 nm (C). (D) U87 cells were treated with G-TPP, harvested at the indicated time intervals, and analyzed by immunoblotting. The position of the unmodified (I) or lipi- dated (II) form of LC3 is indicated. (E) U251 cells were transfected with LC3-GFP, treated with vehicle or G-TPP, and analyzed after 16 hours by fluorescence microscopy (left). The percentage of cells with punctate GFP fluorescence was quantified (right). Mean ± SD of 4–6 independent microscopy fields. (F) U87 cells were incubated with inhibitors of phagosome formation, bafilomycin A (BF), or 3-methyladenine (3-MA) for 1 hour, treated with G-TPP, and analyzed after 16 hours by MTT. Mean ± SD of replicates. (G) L229 cells were transfected with control or atg5- directed siRNA and analyzed by immunoblotting. (H) siRNA-transfected LN229 cells as in G were analyzed for cell viability after 16 hours by MTT. Mean ± SD of replicates.

Article Snippet: Antibodies to LC3 (Cell Signaling), CypD, COX-IV (1:1000; Calbiochem), cytochrome c (1:1000; Clontech), Hsp70 (1:1000; Abcam), TRAP-1 (BD), Ki-67 (Zymed; Invitrogen), caspase-9 (1:250; CST), caspase-8 (1:250; CST), caspase-3 (1:100; CST), caspase-7 (1:500; CST), FLIP-L, long form (1:250; CST) FLIP-S, short form (1:250; CST), CrmA (SCBT), β-actin (1:2000; Sigma-Aldrich), mitochondrial superoxide dismutase-2 (SOD2; 5 μg/ml; Sigma-Aldrich), survivin (NOVUS Biologicals), Bcl-2 (1:1000; CST), Bax (1:1000; CST), PUMA (1:1000; CST), Bcl-XL (1:1000; CST), Bak (1:1000; CST), Bad (1:1000; CST), Bid (1:1000; CST), Mcl-1 (1:1000; CST), C/EBP/β (1:1000; CST), CHOP (1:1000; CST), TAK1 (1:1000; Santa Cruz Biotechnology Inc.), α-glial fibrillary acidic protein (Dako), CD31 (BD Biosciences — Pharmingen) or factor VIII (Dako), or neuronal neurofilament proteins 70, 160, and 200 (all from Progen) were used.

Techniques: Immuno-Electron Microscopy, Western Blot, Transfection, Fluorescence, Microscopy, Incubation, Control

Journal: JCI Insight

Article Title: Elevated CCL2 causes Leydig cell malfunction in metabolic syndrome

doi: 10.1172/jci.insight.134882

Figure Lengend Snippet: ( A and B ) H&E ( A ) and insulin-like 3 (INSL3) ( B ) staining on representative testicular sections; scale bar: 20 μm. ( C and D ) Protein expression of INSL3 ( C ) and STAR ( D ), respectively, as analyzed by Western blot. ( E ) mRNA levels of steroidogenic genes in WT and db/db mice. ( F and G ) Mean testosterone levels in serum ( F ) and testicular interstitial fluid (TIF) ( G ). Data given in C – G were from n = 3–8 mice, 12–24 weeks old, in each group. Data are shown as means ± SEM. Student’s 2-tailed t test was used to compare means between 2 groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Sections were deparaffinized and rehydrated before antigen retrieval, followed by processing for antibody staining against the Leydig cell marker protein INSL3 (orb18041, AA range: 10–50, 1:100, Biorbyt) or the macrophage marker protein F4/80 (MCA497GA, Cl:A3-1, 1:500, AbD).

Techniques: Staining, Expressing, Western Blot

Journal: Nature Communications

Article Title: Lactate mitochondrial oxidation drives stemness potential in metastatic breast cancer

doi: 10.1038/s41467-025-67091-y

Figure Lengend Snippet: A , B Schematic of 2- 2 H-lactate metabolism ( A ) and 2 H-labeled M + 1 metabolites ( B , n = 3 biologically independent samples). ** P = 0.0018, **** P < 0.0001; two-tailed t test. C , D CD147/MCT1/LDHB protein levels in mitochondria ( C ) and sub-compartments ( D ) from AR cells. K proteinase K, T triton X-100, O/I outer membrane and intermembrane space, I/M inner membrane and matrix. COX IV, Na + -K + ATPase, α-Tubulin, TOMM20 mark of inner membrane, plasma membrane, cytoplasm, outer membrane. Data are representative of 3 independent experiments. E Transmission electron microscopy shows CD147/MCT1/LDHB co-localization (orange arrows) in the mitochondrial inner membrane of AR cells. Scale bars: 1 μm (left), 500 nm (right). Red asterisks, mitochondria. F Proximity ligation assay (PLA) between CD147 and LDHB in lactate-treated MCF7 cells. Scale bars, 5 μm. n = 6 typical fields from 3 biologically independent samples. DAPI (blue), PLA signal (red). **** P < 0.0001; two-tailed t test. G Pull-down assay between GST-CD147 N-terminal and His-LDHB. Representative of 3 independent experiments. H CD147 (red)/LDHB (blue)/MCT1 (purple) co-localization (orange arrows) in COX IV (green) labeled mitochondria from PDX1 lung metastatic tumors. Scale bars: 20 μm. I CD147 (green)/LDHB (yellow)/MCT1 (orange) expressions in CK18 (red) labeled cells from breast cancer patients with or without metastasis. Scale bars: 40 μm. J Correlation between CD147 and MCT1/LDHB in 11 metastatic patients with breast cancer. R 2 , Pearson correlation coefficient. Spearman correlation analysis. K Kaplan–Meier survival (up) and recurrence (down) analysis for 131 patients stratified by CD147/MCT1/LDHB co-expression (high n = 9 patients, low n = 31 patients). L Representative images of HE (left: scale bars, 50 μm) and SOX2 IHC staining (middle: scale bars, 50 μm ; right : enlarged image, scale bars, 10 μm) in CTCs from No. 9 patient in Fig. . M Co-localization (orange arrows) of CD147 (red)/LDHB (blue)/MCT1 (purple) in COX IV (green) labeled mitochondria of CTCs from No. 9 patient in Fig. . Scale bars: 20 μm. N SOX2 IHC intensity in CTCs from 10 patients stratified by mitochondrial CD147/MCT1/LDHB complex presence (without, n = 4 patients; with, n = 6 patients). *** P = 0.0004, two-tailed t test. Data are mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: For cellular co-localization analysis among CD147, MCT1, LDHB, and COX IV, cells were initially stained with primary antibodies against CD147 (1:200), MCT1 (1:200; 20139-1-AP, Proteintech), and LDHB (1:100; ab195964, Abcam) overnight at 4 °C, followed by incubation with Alexa Fluor 555-, 647- or 405-conjugated antibodies, respectively, at room temperature for 1 h. This was followed by incubation with an Alexa Fluor 488-conjugated COX IV antibody (1:200; CL488-60251, Proteintech) at room temperature for 3–4 h. Finally, nuclei were counterstained, and images were captured following the same procedures as described above.

Techniques: Labeling, Two Tailed Test, Membrane, Clinical Proteomics, Transmission Assay, Electron Microscopy, Proximity Ligation Assay, Pull Down Assay, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Lactate mitochondrial oxidation drives stemness potential in metastatic breast cancer

doi: 10.1038/s41467-025-67091-y

Figure Lengend Snippet: A Immunoactivity of EGFP-fused mito-CD147 scFv to CD147 in MCF7/T47D cell subfractions: C (plasma membrane and cytoplasm), M (mitochondria), COX IV, α-Tubulin mark mitochondrial inner membrane, cytoplasm. NC: pGV469 vector; scFv: pGV469-mito-CD147 scFv. Representative of 3 independent experiments. B Co-IP assays showing CD147 interaction with MCT1/LDHB in mitochondria from lactate-treated MCF7 WT/KO/mito147 cells as indicated. Groups: 1. WT; 2. WT+mito-CD147 scFv; 3. KO; 4. KO+mito-CD147 scFv; 5. mito147; 6. mito147+mito-CD147 scFv. Representative of 3 independent experiments. C–F Experimental design ( C ), lung nodules ( D , n = 10 mice/group), ALDH + and CD44 high CD24 −/low ratios ( E , n = 5 mice/group), and SOX2 levels ( F , n = 4 mice/group, α-Tubulin, loading control) in tumors from MMTV-PyMT mice as indicated. *** P = 0.0001 ( E left), *** P = 0.0004, 0.0003 ( E right, NS vs Lac, Lac vs Lac + scFv), **** P < 0.0001, one-way ANOVA with Bonferroni correction. C created in BioRender. Song, C. (2025) https://BioRender.com/37z58my . G , H CTC counts ( G , left, n = 8 mice/group), SOX2 intensity ( G , right; NS, n = 148 cells from 7 mice; Lac, n = 342 cells from 7 mice; Lac+scFv, n = 149 cells from 7 mice), and representative images of CTCs ( H ; scale bars, 40 μm), from MMTV-PyMT mice as indicated. H DAPI (blue), CK18 (green), SOX2 (purple). **** P < 0.0001, one-way ANOVA with Bonferroni correction. I , J Lung nodules ( I , n = 8 mice/group), ALDH + and CD44 high CD24 −/low ratios ( J , n = 5 mice/group) in tumors from PDX1-bearing mice as indicated. ns, P = 0.9818, 0.869 ( J left, right), *** P = 0.0001, **** P < 0.0001, one-way ANOVA with Bonferroni correction. K–M CTC counts ( K , left, n = 8 mice/group), SOX2 intensity ( K , right; NS, n = 80 cells from 8 mice; scFv, n = 80 cells from 8 mice; Lac, n = 120 cells from 6 mice; Lac+scFv, n = 80 cells from 8 mice), representative CTC images ( L ), and mito-CD147 scFv binding to mitochondrial CD147 ( M ) in CTCs from PDX1-bearing mice as indicated. Scale bars: 10 μm. L DAPI (blue), CK18 (green), SOX2 (purple). M DAPI (blue), CD147 (red), scFv (green), COX IV (purple). CTC circulating tumor cell, scFv single-chain variable fragment. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: For cellular co-localization analysis among CD147, MCT1, LDHB, and COX IV, cells were initially stained with primary antibodies against CD147 (1:200), MCT1 (1:200; 20139-1-AP, Proteintech), and LDHB (1:100; ab195964, Abcam) overnight at 4 °C, followed by incubation with Alexa Fluor 555-, 647- or 405-conjugated antibodies, respectively, at room temperature for 1 h. This was followed by incubation with an Alexa Fluor 488-conjugated COX IV antibody (1:200; CL488-60251, Proteintech) at room temperature for 3–4 h. Finally, nuclei were counterstained, and images were captured following the same procedures as described above.

Techniques: Clinical Proteomics, Membrane, Plasmid Preparation, Co-Immunoprecipitation Assay, Control, Binding Assay