Journal: Cell metabolism

Article Title: Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans

doi: 10.1016/j.cmet.2005.09.002

Figure Lengend Snippet: Preconfluent myoblasts were transiently transfected with pcDNA3.0-hSCD1 or the empty parent vector. After 7 days in DFM, cells were harvested to obtain total RNA. Overexpression of SCD1 was confirmed in differentiated myotubes by (A) semiquantitative PCR of mRNA and (B) Western blot analysis of protein. β-actin mRNA and GAPDH protein were used as loading controls. Metabolic assays were performed following a 3 hr incubation with 100 μM [14C]oleate and 0.25% BSA. (C) Fatty acid oxidation was determined by measuring 14C-label incorporation into CO2. (D) Glycerolipid synthesis was determined by measuring 14C-label incorporation into triacylglycerol (TAG) and phospholipid (PL). All assays were performed in triplicate and values represent the mean ± SEM from four separate experiments. Differences between groups were analyzed by Student’s t test, *p < 0.05, **p < 0.01. (E) ACCβ mRNA levels were quantified by RTQ-PCR and are expressed as relative units in cells transfected with SCD1 versus empty vector controls. Cyclophilin B was measured as an endogenous control. Data are representative of five experiments and differences between groups were analyzed by Student’s t test, *p < 0.05. (F) Total and phosphorylated AMPK and ACCβ were analyzed by immunoblotting with antibodies against the AMPKα 1 and 2 catalytic subunits, ACCβ, AMPK-thr172, and ACCβ-ser79. GAPDH protein expression was not different between groups.

Article Snippet: Blots were probed with primary antibodies against hSCD1 (4 μg/mL, Alpha Diagnostics International, San Anto-nio, TX) and GAPDH (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) followed by anti-rabbit and anti-goat secondary antibodies respectively (1:8000 and 1:10000 dilutions, respectively; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Over Expression, Western Blot, Incubation, Control, Expressing

Journal: Pain

Article Title: Peripheral oxytocin restores light touch and nociceptor sensory afferents towards normal after nerve injury

doi: 10.1097/j.pain.0000000000001495

Figure Lengend Snippet: A. Representative vasopressin 1A receptor immunoreactivity in sections from L4 dorsal root ganglia (DRGs) visualized by fluorescence microscopy (vasopressin 1a receptors in green and NeuN in red). Calibration bar: 50 μm.

Article Snippet: The anti-vasopressin 1A receptor antibody (rabbit, 1:100, #AVP1A11-P, Alpha Diagnostics, San Antonio, TX, USA) showed selective immunostaining in brain regions of vasopressin 1A receptor location in the rat brain and consistent immunostaining in a subset of dorsal root ganglion neurons and was used for tissues from animals in the experiment.

Techniques: Fluorescence, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Byakangelicin protects against carbon tetrachloride–induced liver injury and fibrosis in mice

doi: 10.1111/jcmm.15493

Figure Lengend Snippet: Byakangelicin treatment reduces carbon tetrachloride‐induced liver fibrosis. A, Animal liver fibrosis modelling plan. B, Detection of hydroxyproline in liver tissue. C, Classical tissue staining in liver fibrosis: Sirius red, Masson and α‐SMA staining; D, three stained quantitative areas. E, Protein content of α‐SMA and COL‐1 in liver tissue; F, corresponding grey analysis. G, Real‐time PCR analyses of α‐SMA and COL‐1 in liver tissues. For the statistics of each panel in this figure, * P < .05 vs model, ** P < .01 vs model, *** P < .001 vs model, n = 3

Article Snippet: Primary antibodies including anti‐α‐smooth muscle actin (α‐SMA), anti‐collagen I (COL‐1), anti‐GAPDH and anti‐p‐Stat3, Stat3, β‐tubulin, cyclin D1, P53, p‐ASK‐1, ASK‐1, IL‐1β, NF‐κB and Nrf‐2 were acquired from Affinity Biosciences; anti‐p‐Smad3, Smad3, p‐PDGFR, PDGFR, p‐ERK, ERK, p‐AKT, AKT, PARP, cleaved caspase‐3 and caspase‐3 were received from (Cell Signaling Technology); anti‐p‐JNK, JNK from Signalway Antibody LLC; and anti‐4‐HNE from Abcam.

Techniques: Staining, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Byakangelicin protects against carbon tetrachloride–induced liver injury and fibrosis in mice

doi: 10.1111/jcmm.15493

Figure Lengend Snippet: Byakangelicin inhibits the TGF/Smad3 pathway in TGF‐β‐induced activation of hepatic stellate cell. A, Detection of byakangelicin's cytotoxicity in liver stellate cell line LX2 using MTT. B, Real‐time PCR analyses of α‐SMA and COL‐1 in LX2 cells. C and D, Western blot analyses of α‐SMA, COL‐1, P‐Smad3, Smad3 and GAPDH protein expression in LX2 cells with densitometry. E and F, Immunofluorescence using antibody against α‐SMA and Smad3. For the statistics of each panel in this figure, * P < .05, ** P < .01, *** P < .001, n = 3

Article Snippet: Primary antibodies including anti‐α‐smooth muscle actin (α‐SMA), anti‐collagen I (COL‐1), anti‐GAPDH and anti‐p‐Stat3, Stat3, β‐tubulin, cyclin D1, P53, p‐ASK‐1, ASK‐1, IL‐1β, NF‐κB and Nrf‐2 were acquired from Affinity Biosciences; anti‐p‐Smad3, Smad3, p‐PDGFR, PDGFR, p‐ERK, ERK, p‐AKT, AKT, PARP, cleaved caspase‐3 and caspase‐3 were received from (Cell Signaling Technology); anti‐p‐JNK, JNK from Signalway Antibody LLC; and anti‐4‐HNE from Abcam.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Byakangelicin protects against carbon tetrachloride–induced liver injury and fibrosis in mice

doi: 10.1111/jcmm.15493

Figure Lengend Snippet: Byakangelicin inhibits the proliferation and activation of PDGF‐induced hepatic stellate cell by inhibiting PDGFR/ERK, PDGFR/AKT, and PDGFR/Stat3. A and B, Western blot analyses of P‐PDGFR, PDGFR, P‐ERK, ERK, P‐AKT, AKT, P‐Stat3, Stat3, α‐SMA and cyclin D1 protein expression with densitometry. C and D, Immunofluorescence by using antibody against cyclin D1 and PDGFR. For the statistics of each panel in this figure, * P < .05, ** P < .01, *** P < .001, n = 3

Article Snippet: Primary antibodies including anti‐α‐smooth muscle actin (α‐SMA), anti‐collagen I (COL‐1), anti‐GAPDH and anti‐p‐Stat3, Stat3, β‐tubulin, cyclin D1, P53, p‐ASK‐1, ASK‐1, IL‐1β, NF‐κB and Nrf‐2 were acquired from Affinity Biosciences; anti‐p‐Smad3, Smad3, p‐PDGFR, PDGFR, p‐ERK, ERK, p‐AKT, AKT, PARP, cleaved caspase‐3 and caspase‐3 were received from (Cell Signaling Technology); anti‐p‐JNK, JNK from Signalway Antibody LLC; and anti‐4‐HNE from Abcam.

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Viral-like TLR3 induction of cytokine networks and α-synuclein are reduced by complement C3 blockade in mouse brain

doi: 10.1038/s41598-023-41240-z

Figure Lengend Snippet: Intra-striatal TLR3 activation in mouse brain increased complement C3 and α-synuclein protein levels. WT mice received a single unilateral deposit of 50 μg Poly(I:C) (n = 10) or vehicle (n = 9) in the striatum. Two days later, injected striata were isolated and homogenised in RIPA buffer for protein detection by Western blot ( A, E ). ( B – D, F – I ) Histogram bars represent the mean levels of target protein normalized to GAPDH or total protein visualized by stain-free UV-fluorescence. Uncropped blots can be found in Figs. – . Error bars represent SEM. A.U., arbitrary units. Statistical analysis was performed using unpaired t-tests. ns, p > 0.05; *, p < 0.05; ***, p < 0.001.

Article Snippet: Membranes were blocked for 30 min with 5% milk in Tris-buffered saline containing 0.1% Tween 20 and were probed overnight at 4 °C with primary antibodies against the following target proteins: α-synuclein (BD biosciences, BD610787 1:2000), C3 (Abcam, ab200999, 1:1000), C1q (Abcam, ab182451, 1:500), Iba1 (Abcam, ab178846, 1:1000), GFAP (EMD Millipore, MAB360, 1:2000), GAPDH (Cell Signalling Technology, #2118, 1:2000).

Techniques: Activation Assay, Injection, Isolation, Western Blot, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Viral-like TLR3 induction of cytokine networks and α-synuclein are reduced by complement C3 blockade in mouse brain

doi: 10.1038/s41598-023-41240-z

Figure Lengend Snippet: Knockdown of C3 prior to TLR3 activation reduced α-synuclein protein levels in the striatum of mouse brain. Alpha-synuclein levels in the injected striatum of mice from NT ASO/Poly(I:C) (n = 10) and C3 ASO/Poly(I:C) (n = 10) groups were measured by Western blot ( A ). The uncropped blot can be found in Fig. . ( B ) Histogram bars represent the mean levels of α-synuclein normalized to total protein loading. Error bars represent SEM. A.U., arbitrary units. Statistical analysis was performed using unpaired t-tests. ***, p < 0.001.

Article Snippet: Membranes were blocked for 30 min with 5% milk in Tris-buffered saline containing 0.1% Tween 20 and were probed overnight at 4 °C with primary antibodies against the following target proteins: α-synuclein (BD biosciences, BD610787 1:2000), C3 (Abcam, ab200999, 1:1000), C1q (Abcam, ab182451, 1:500), Iba1 (Abcam, ab178846, 1:1000), GFAP (EMD Millipore, MAB360, 1:2000), GAPDH (Cell Signalling Technology, #2118, 1:2000).

Techniques: Activation Assay, Injection, Western Blot

Journal: Redox Biology

Article Title: Involvement of the mitochondrial nuclease EndoG in the regulation of cell proliferation through the control of reactive oxygen species

doi: 10.1016/j.redox.2020.101736

Figure Lengend Snippet: Analysis of the impact of Endog expression in cell proliferation and ROS abundance in diverse mouse, rat and human cell types. A) Cardiomyocyte (CM) number was counted from 5 to 9 hearts of Endog +/+ and Endog −/− of 4-days-old (P4) male and female mice (representative images shown at right), after digestion of the ventricular tissue that was previously weighted (B). C) Quantification of the expression of the proliferation-related Ki-67 nuclear antigen in histological ventricular tissue preparations from P4 Endog +/+ and Endog −/− mice. Ki-67+ nuclei were counted in 3 different slices from 3 hearts/genotype and data are expressed as Ki-67 + nuclei/total nuclei in the slice. All individual quantifications are plotted (1000–2000 nuclei/genotype). D) MitoSOX™ fluorescence was quantified by flow cytometry in preparations of ventricular cardiomyocytes from P4 Endog +/+ and Endog −/− mice cultured in the presence or absence of 0.2 mM N -Acetyl-Cysteine (NAC; n = 6). E) Neonatal rat ventricular cardiomyocyte cultures were left untreated (NT, not transduced), transduced with a scrambled sequence (Scr) or an Endog -specific silencing sequence (shRNA) and then treated or not with 0.2 mM NAC for 48 h. Cells were fixed and stained with muscle-specific α-actinin (cardiomyocytes) and Hoechst dye (nuclei). Cardiomyocytes were counted in 10 different microscopic fields/treatment in 4 independent experiments. All 40 values/treatment are plotted. F) Skin fibroblasts were obtained from P4 Endog +/+ and Endog −/− mice, plated and amplified. Equal number of cells were seeded in 2 plates/genotype and counted after 72 h. Data are expressed as the number of cell cycles completed in 72 h.Cells in replicate plates were counted at time zero to confirm equal initial cell number. All values from 4 independent experiments are plotted. G) MitoSOX™ fluorescence was quantified as in (D) in preparations of Endog +/+ and Endog −/− fibroblasts treated or not with NAC. H) Endog +/+ and Endog −/− fibroblasts were seeded in presence or absence of NAC and counted after 48 h. Conditions are as in F. I) Strategy for the CRISPR-Cas9-dependent ENDOG gene truncation by insertion of the puromycin resistance cassette (PuroR), using homologous recombination in 4 different selected sites within the ENDOG gene in HEK293 human cells. Expression of ENDOG was analyzed in control cells (C) and several clones from each of the four PuroR insertion sites (see Material and methods section). J) MitoSOX™ fluorescence was quantified as in (D) in preparations of control cells and cells from CRISPR-Cas9-treated clones 1.1 and 4.3 treated or not with 0.2 mM NAC (n = 7). K) HEK293 cells were counted in control and ENDOG-deficient cultures from clones 1.1 and 4.3 cultured for 48 h, treated or not with NAC. Proliferation was expressed as cell cycles completed in 48 h. Data plotted are from four experiments performed in duplicates. Graphs show experimental values plus mean ± SD, except D, G and J where median ± interquartile range is depicted. Analysis of the effect of Endog expression in the experimental values was performed with the Student's t-test (B, C and F). 2-way ANOVA was used to analyze the influence of Endog expression and gender (A) or NAC treatment (E, H and K) and the interaction between them in cell number. The Kruskal-Wallis test was used to compare the effects of Endog expression and NAC addition on MitoSOX™ fluorescence followed by the Dunn's test for selected post hoc comparisons (D, G and J). ns = not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Immunohistochemistry was performed in paraffin-included heart slices as previously described [ ] using primary antibodies against Ki67 (MAB4190, Merck) and Cyclin D (SC-20044), biotinylated secondary antibodies (Jackson) and developed with deaminobenzidine (DAB, DAKO) contrasted with hematoxylin.

Techniques: Expressing, Fluorescence, Flow Cytometry, Cell Culture, Transduction, Sequencing, shRNA, Staining, Amplification, CRISPR, Homologous Recombination, Clone Assay