Journal: Cancer Cell International

Article Title: Darunavir analog precursors target mitochondrial metabolism in multiple myeloma and CLL.

doi: 10.1186/s12935-025-04142-w

Figure Lengend Snippet: The HIV protease inhibitors and BnpM-NH 2 affect the respiratory chain function altering the the expression of complex II and IV proteins . ( A ) WB analysis of NDUFB8, SDHB, UQCRC2, MTCO2, ATP5A1, and Actin expression in MM1S, RPMI-8226, and HG3 cells treated with BupM-NH2 and BnpM-NH2 (30 µM) for 48 h was performed. The intensity of NDUFB8, SDHB, UQCRC2, MTCO2, ATP5A1, and Actin intensity normalized to actin were quantified and plotted as bar graphs. The results are presented as mean ± SD of at least three replicates. ( B ) ROS production and mitochondrial mass in MM and CLL cells treated with BupM-NH 2 and BnpM-NH 2 (30 µM) for 24 h were assessed using mitosox and mitotracker assays. FACS analysis was employed to detect median fluorescent intensity (MFI), which was reported on a bar graph. Data, presented as mean ± SD, were obtained from at least three independent experiments. Significant differences ( P ≤ 0.05) were determined by one-way ANOVA with Tukey’s post analysis

Article Snippet: MTCO2 (1:6000, 26 KDa) , Proteintech , Cat#: 55070-1-AP; RRID: AB_10859832.

Techniques: Expressing

Journal: Cancer Discovery

Article Title: Copper Drives Remodeling of Metabolic State and Progression of Clear Cell Renal Cell Carcinoma

doi: 10.1158/2159-8290.CD-24-0187

Figure Lengend Snippet: Cu-dependent activation of CuCOX promotes oxygen consumption, reorganization of respiratory supercomplexes, and nucleotide biosynthesis essential for Cu-dependent tumor growth. A–D, OCR from representative Seahorse mitochondrial stress tests in 786-O ( A and B ) and RCC4 ( C and D ) cells. Quantification of basal respiration, maximal respiration (post-FCCP injection), and respiration coupled to ATP production. P value from paired t test. E, Blue NativePAGE (BN-PAGE) analysis of respiratory complexes using digitonin permeabilized mitochondria isolated by anti-TOM22 immunopurification from Cu Hi vs. Cu Lo cells. Immunoblotting for indicated respiratory complex subunits. Red brackets indicate RSC. F, In-gel activity assay (IGA) for cytochrome c oxidase. RSCs are indicated with red brackets. G, Quantification of Cu Hi complex IV (COX) IGA relative to Cu Lo . P values are calculated by a one-sample t test. H, Western blot for COX subunits MT-CO1 and MT-CO2 and RSC assembly factor COX7A2L in mitochondrial lysates. I, BN-PAGE of mitochondria shows enrichment for COX7A2L in RSCs (red bracket) in Cu Hi cells. J, Quantification of COX7A2L in western blots of RSCs shown in M . P values calculated by one-sample t test. K, BN-PAGE of mitochondria shows enrichment for COX17, chaperone of Cu to CuCOX, in respiratory complexes from Cu Hi cells. L, Total CL content measured using mitochondrial lipidomics in 786-O and RCC4 cells. RA, relative abundance. M, Total CL content in xenografts formed by 786-O cells or in XP374d tumors in mice fed low and high Cu diet. N, Cu effects on labeling of 6-PDG, intermediate of oxidative branch of PPP, after 5 hours of incubation with [ 13 C 6 ]-glucose. O, Fractional enrichment of [ 13 C 5 ]- nucleotides labeled from [ 13 C 6 ]-glucose in a total pool of each nucleotide in Cu Lo and Cu Hi cells after 24 hours of incubation. P, Fractional enrichment of nucleotides labeled from [ 13 C 5 , 15 N 2 ]-glutamine in a total pool of each nucleotide in Cu Lo and Cu Hi cells after 24 hours of incubation. Q, Gross images of tumors formed by control 786-O cells expressing nontargeting (NT) or cells with COX4I1 knockdown in mice fed matched Cu Lo or Cu Hi diets. Scale bar, 1 cm. R, Weight of tumors shown in Q at collection. P values were calculated by one-way ANOVA with the Holm–Šidák posttest. S, Gross images of XP374d tumors in mice fed with a Cu Hi diet treated with IACS-10579 or vehicle (V). Scale bar, 1 cm. T, Volume of subcutaneous tumors at the indicated time points. U, The weight of tumors shown in S at collection. V, Representative images of staining for MT-CO2 in noninvasive and invasive orthotopic xenografts. K, kidney tissue; LD, lipid droplets; M, muscle; T, tumor tissue. The dashed line indicates the boundary between the tumor and kidney tissue. Scale bars, 200 μm. Means ± SEM are shown; P values were calculated from unpaired two-tailed t test unless indicated. See also Supplementary Fig. S2 and Supplementary Tables S3, S4A, and S4B.

Article Snippet: The following antibodies were used: Ki-67 (Roche Ventana, 05278384001, RRID: AB_2631262 or Santa Cruz Biotechnology, sc-23900, RRID: AB_627859) and MT-CO2 (1:100, Novus, NBP3-16283, RRID: AB_3599404).

Techniques: Activation Assay, Injection, Isolation, Immu-Puri, Western Blot, Activity Assay, Labeling, Incubation, Control, Expressing, Knockdown, Staining, Two Tailed Test

Journal: Biological Research

Article Title: hUC-MSC preserves erectile function by restoring mitochondrial mass of penile smooth muscle cells in a rat model of cavernous nerve injury via SIRT1/PGC-1a/TFAM signaling

doi: 10.1186/s40659-024-00578-y

Figure Lengend Snippet: MSC restore mitochondrial biogenesis and function via SIRT1/PGC-1a/TFAM pathway. A Immunoblot analysis of protein level of SIRT1 expression in CCSMCs after SIRT1 knockdown by siRNA. β-Actin expression levels were used as an internal control. Full-length blots of CCSMCs were presented (Additional file : Fig. S7). B Immunoblot analysis of protein levels of SIRT1, PGC-1α, TFAM in CCSMCs of different groups. Full-length blots of CCSMCs were presented (Additional file : Fig. S8). C Quantification of relative protein levels of SIRT1, PGC-1α, TFAM levels in different groups. D Immunoprecipitation analysis of acetylation level of PGC-1α in CCSMCs. Full-length blots of CCSMCs were presented (Additional file : Fig. S8). E Quantification of acetylation level of PGC-1α in different groups. Acetylation level of PGC-1α were normalized to PGC-1α. F Representative images of mitochondria of CCSMCs stained with MitoTracker deep red (red) or anti-TOM20 monoclonal antibody (green). Scale bar: 20 µm. G Quantification of relative fluorescence intensity of MitoTracker deep red or TOM20 of per cell in CCSMCs in different groups. At least 20 cells were counted for each group and data are presented as mean ± SD. H Quantification of the mtDNA copy number in CCSMCs. Data were collected from at least three independent experiments, and the data are presented as mean ± SD. I Intracellular ATP level in CCSMCs after knockdown of SIRT1 and coculture with hUC-MSCs. Data were collected from at least three independent experiments and data are presented as mean ± SD. J Immunoblot analysis of protein levels of NDUFB8, SDHB, UQCRC2, MTCO2 and ATP5A in CCSMCs in different groups. Full-length blots of CCSMCs were presented (Additional file : Fig. S10). K Quantification of relative protein levels of NDUFB8, SDHB, UQCRC2, MTCO2 and ATP5A levels of CCSMCs in different groups. L Flow cytometry analysis of Annexin V / PI staining of CCSMCs in different groups. M Quantification of apoptotic CCSMCs in different groups. Data were collected from at least independent groups, and the data are presented as mean ± SD. N Immunoblot analysis of protein levels of Cleaved caspase-3, Caspase-3 and Bcl-2 protein in CCSMCs. Full-length blots were presented (Additional file : Fig. S11). O Quantification of relative protein levels of Cleaved caspase-3 and Bcl-2 levels in CCSMCs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: The information of the primary antibodies is as follows: SIRT1 (Abcam), TOM20 (Abcam), PGC-1a (Novus Biologicals), MTCO2, UQCRC2, SDHB, NDUFB8, ATP5A, β-Actin (above all from Proteintech), Cleaved caspase3, Caspase3, Bcl2 (above all from Abclonal).

Techniques: Western Blot, Expressing, Knockdown, Control, Immunoprecipitation, Staining, Fluorescence, Flow Cytometry