Journal: Biomedicines

Article Title: The Identification of Prohibitin in the Rat Heart Mitochondria in Heart Failure

doi: 10.3390/biomedicines9121793

Figure Lengend Snippet: Effects of the administration of AX on the content of PHB, ANT2, VDAC1, and VDAC2 in rat heart mitochondria in heart failure. Upper part—immunostaining with PHB, ANT2, VDAC1, VDAC2, and Tom20 antibodies; Low part—quantification of immunostaining by computer-assisted densitometry presented as a ratio of proteins to Tom20. Bar graphs represent the levels of appropriate proteins; the data are presented as the means ±SD of three independent experiments. * p < 0.05 significant difference in the protein level in comparison with the control (group 1). # p < 0.05 compared to RHM isolated from ISO-injected rats (group 3). The statistical significance of the differences between the pairs of mean values was evaluated using the Student-Newman-Keul test.

Article Snippet: The polyclonal Tom20 (Santa-Cruz, Dallas, TX, USA) and monoclonal GAPDH (Cell Signaling, Danvers, MA, USA) antibodies were used as a loading control.

Techniques: Immunostaining, Comparison, Control, Isolation, Injection

Journal: Biomedicines

Article Title: The Identification of Prohibitin in the Rat Heart Mitochondria in Heart Failure

doi: 10.3390/biomedicines9121793

Figure Lengend Snippet: Effects of the administration of AX on the content of ATPB, ATP5F1, ATP5G, pGSK3β and GSK3β in rat heart mitochondria in heart failure. ( a )—immunostaining with ATPB, ATP5F1, ATP5G, and Tom20 antibodies (upper part); low part—quantification of immunostaining by computer-assisted densitometry presented as a ratio of proteins to Tom20; ( b )—immunostaining with pGSK3β, GSK3β, and Tom20 antibodies (upper part); low part—quantification of immunostaining by computer-assisted densitometry presented as a ratio of proteins to Tom20. Bar graphs represent the levels of appropriate proteins; the data are presented as the means ± SD of three independent experiments. * p < 0.05 significant difference in the protein level in comparison with the control (group 1). # p < 0.05 compared to RHM isolated from ISO-injected rats (group 3). The statistical significance of the differences between the pairs of mean values was evaluated using the Student-Newman-Keul test.

Article Snippet: The polyclonal Tom20 (Santa-Cruz, Dallas, TX, USA) and monoclonal GAPDH (Cell Signaling, Danvers, MA, USA) antibodies were used as a loading control.

Techniques: Immunostaining, Comparison, Control, Isolation, Injection

Journal: bioRxiv

Article Title: Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

doi: 10.1101/2025.05.01.651794

Figure Lengend Snippet: ( a, b) Age-related decrease in MITOL expression. Western blot analysis of MITOL expression in the heart at each indicated month of age. Signal intensities were normalized to Tubulin and presented as values relative to the 1-month group. (c) Schematic representation of the compound evaluation strategy. Various herbal extracts and previously reported mitochondria-activating bioactive compounds were individually administered to human dermal fibroblasts. Candidate compounds identified based on MITOL mRNA upregulation by qRT-PCR were subsequently administered to mice to assess MITOL expression in multiple organs. (d) Relative MITOL mRNA levels in human dermal fibroblasts after 24-hour treatment with each compound, quantified by qRT-PCR. mRNA levels were normalized to GAPDH and expressed as a percentage relative to the control group (without test substances). Data represent the average of three independent experiments (n = 3). (e, f) Increased MITOL expression in mouse tissues following herbal extract administration. Mice were administered each herbal extract ( Phellodendron amurense , Coptis japonica , or Paeonia suffruticosa ) intraperitoneally, and 9 days later, MITOL expression levels in various organs were assessed by Western blot. f shows the graph of MITOL expression levels in the heart, normalized to tubulin (n = 5– 6 per group). Paeonia suffruticosa extract was included based on its prior effect on MITOL expression in hair follicle keratinocytes (data not shown); however, it did not increase MITOL expression in the heart, liver, or kidney in this experiment. (g, h) Upregulation of MITOL and mitochondrial proteins in C2C12 myoblasts following berberrubine treatment. C2C12 cells were treated with berberine (10 μM) or quinoid-type berberrubine (10 μM) for 48 hours, and protein expression levels were analyzed by Western blot ( g ). h shows the quantification of Western blot signals for MITOL, Tom20, Mfn2, HSP60, and ATP5a, normalized to Vinculin and expressed as values relative to the None group (n = 4 per group). The None group indicates cells cultured without any additives, including solvents. Statistical significance was determined using two-way ANOVA followed by Bonferroni post hoc test (f) or one-way ANOVA followed by Tukey’s HSD test (h) . * p < 0.05; ** p < 0.01; *** p < 0.001; ** p < 0.0001; ns, not significant.

Article Snippet: The following antibodies were used: anti-MITOL (LS-C164034, LSBio); anti-α-Tubulin (T-9026, Sigma-Aldrich); anti-Mfn2 (sc-100560, Santa Cruz Biotechnology); anti-HSP60 (sc-136291, Santa Cruz Biotechnology); anti-ATP5a (ab14748, Abcam); anti-Tom20 (11802-1-AP, Proteintech); and anti-Vinculin (V9131, Sigma-Aldrich).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Cell Culture

Journal: bioRxiv

Article Title: Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

doi: 10.1101/2025.05.01.651794

Figure Lengend Snippet: (a) C2C12 cells were treated with Q-BRB or AcA-BRB (5 μM, 10 μM), and the protein levels of mitochondrial-localized proteins (outer membrane: MITOL, Tom20, Mfn2; matrix: HSP60; inner membrane: ATP5a) were detected by Western blotting. Signal intensities were normalized to Vinculin and expressed as values relative to the 0 μM group (n = 4 per group). The 0 μM group indicates cells cultured without any additives, including solvents. (b) mRNA levels of MITOL and Tom20 were measured by qRT-PCR and normalized to GAPDH and expressed as fold change relative to the 0 μM group (n = 3 per group). (c) Mitochondrial content was quantified by real-time PCR based on the ratio of mtDNA (16S rRNA or ND1) to nuclear DNA (Hexokinase 2; HK2) and expressed as fold change relative to the 0 μM group (n = 3 per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s HSD test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The following antibodies were used: anti-MITOL (LS-C164034, LSBio); anti-α-Tubulin (T-9026, Sigma-Aldrich); anti-Mfn2 (sc-100560, Santa Cruz Biotechnology); anti-HSP60 (sc-136291, Santa Cruz Biotechnology); anti-ATP5a (ab14748, Abcam); anti-Tom20 (11802-1-AP, Proteintech); and anti-Vinculin (V9131, Sigma-Aldrich).

Techniques: Membrane, Western Blot, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Mitorubin, berberrubine-based compounds that improve mitochondrial function, exhibit cardioprotective effects against age-related cardiac dysfunction

doi: 10.1101/2025.05.01.651794

Figure Lengend Snippet: (a) C2C12 cells were treated with Q-BRB or AcA-BRB (10 μM) for 48 h, followed by immunostaining with an anti-Tom20 antibody to visualize mitochondrial morphology (green: anti-Tom20; blue: Hoechst). Representative images are shown in the upper panels. Enlarged views of the boxed regions are displayed in the lower panels. Scale bars: 20 μm (upper), 10 μm (lower). The None group indicates cells cultured without any additives, including solvents. (b) Mitochondrial length was quantified in 30 randomly selected cells per group using MiNA (Mitochondrial Network Analysis). Mitochondria were classified into four categories based on length: Short (≤1.855 μm), Middle (1.855–3.599 μm), Long (3.599–8.103 μm), and Very long (≥8.103 μm), and presented as the proportion of mitochondria in each category within each group. Representative images for each category are shown on the right. Scale bar: 10 μm. (c, e) C2C12 cells were treated with Q-BRB or AcA-BRB (30 μM) for 24 h, and mitochondrial oxygen consumption rate (OCR) was measured (n = 3 per group). (d, f) Basal respiration, ATP synthesis-linked respiration, and maximal respiration were calculated from the OCR data obtained in (c, e) after sequential administration of oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Statistical significance was determined by comparing each treatment group (Q-BRB or AcA-BRB) with the corresponding solvent control group (DMSO or H 2 O) using a two-sided unpaired Student’s t -test. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were used: anti-MITOL (LS-C164034, LSBio); anti-α-Tubulin (T-9026, Sigma-Aldrich); anti-Mfn2 (sc-100560, Santa Cruz Biotechnology); anti-HSP60 (sc-136291, Santa Cruz Biotechnology); anti-ATP5a (ab14748, Abcam); anti-Tom20 (11802-1-AP, Proteintech); and anti-Vinculin (V9131, Sigma-Aldrich).

Techniques: Immunostaining, Cell Culture, Solvent, Control

Journal: Antioxidants

Article Title: The Effect of Astaxanthin on Mitochondrial Dynamics in Rat Heart Mitochondria under ISO-Induced Injury

doi: 10.3390/antiox12061247

Figure Lengend Snippet: The influence of AX and ISO change of DRP1, Mfn2, and OPA1 in RHM. ( a ) upper part—Western blot with antibody to DRP1. Tom20 was used for normalization of proteins. Lower part—quantitative characteristic presented as the ratio of DRP1 to Tom20; ( b ) upper part—Western blot with antibody to Mfn2, lower part—quantitative characteristic presented as the ratio of Mfn2 to Tom20; ( c ) upper part—Western blot with antibody to OPA1, lower part—quantitative characteristic presented as the ratio of OPA1 to Tom20. The data are presented as the means ± SDs of four independent experiments. * p < 0.05 significant values compared with control (group 1); # p < 0.05 significant values relative ISO to (group 3).

Article Snippet: The polyclonal anti-Tom20 antibodies (Cell Signaling, Danvers, MA, USA) at dilution 1:1000 were used to normalize the protein.

Techniques: Western Blot

Journal: Antioxidants

Article Title: The Effect of Astaxanthin on Mitochondrial Dynamics in Rat Heart Mitochondria under ISO-Induced Injury

doi: 10.3390/antiox12061247

Figure Lengend Snippet: The influence of AX and ISO change of PHB2 and PINK1 in RHM. ( a ) upper part—Western blot with antibody to PHB2. Tom20 was used for normalization of protein. Lower part—quantitative characteristic presented as the ratio of PHB2 to Tom20; ( b ) upper part—Western blot with antibody to PINK1, lower part—quantitative characteristic presented as the ratio of PINK1 to Tom20. The data are presented as the means ± SDs of four independent experiments. * p < 0.05 significant values compared with control (group 1); # p < 0.05 significant values relative to ISO (group 3).

Article Snippet: The polyclonal anti-Tom20 antibodies (Cell Signaling, Danvers, MA, USA) at dilution 1:1000 were used to normalize the protein.

Techniques: Western Blot