Journal: Cancers

Article Title: CPT1A Over-Expression Increases Reactive Oxygen Species in the Mitochondria and Promotes Antioxidant Defenses in Prostate Cancer

doi: 10.3390/cancers12113431

Figure Lengend Snippet: Mitochondrial reactive oxygen species (ROS) are increased in CPT1A-OE cells. ( A ) Electron paramagnetic resonance spectroscopy (EPR) traces of the four cell lines tested, including the mitochondrial probe by itself. Amplitude and linewidth provide information of the amount of ROS. The concentration was acquired by Spin Fit followed by SpinCount module (Bruker). ( B ) Quantification of the EPR traces normalized to protein content. *** p < 0.001. ( C ) Gene expression analysis of OE versus KD cells for SOD1 ns), SOD2 ( p = 6.9 × 10 −4 ), and SOD3 ( p = 0.015). Only SOD2 is a mitochondrial enzyme. ( D , E ) Western blots of SOD2 expression after incubation with fatty acids (Oleic and palmitate mixture; 25 µM each) in FBS ( D ) or charcoal stripped serum (CSS) ( E ) for 48 h. ( F ) Schematic of the role of SOD2 in metabolizing superoxide and the role of glutathione in eliminating H 2 O 2 . GPX = Glutathione peroxide. GSSG = oxidized glutathione.

Article Snippet: Antibodies: CPT1A: 15184-1-AP, (Proteintech, Rosemont, IL, USA); GAPDH: CST 5174, (Cell Signaling Technology, Beverly, MA, USA); SOD2: CST 13141, Cell Signaling Technology.

Techniques: Electron Paramagnetic Resonance, Spectroscopy, Concentration Assay, Expressing, Western Blot, Incubation

Journal: Journal of Advanced Research

Article Title: Neural and molecular investigation into the paraventricular thalamus for chronic restraint stress induced depressive-like behaviors

doi: 10.1016/j.jare.2024.10.025

Figure Lengend Snippet: CRS induced depressive-like behaviors and reduced the activity of PVT CamkIIα neurons in mice. (A) Experimental paradigm for chronic stress restraint. (B) tail suspension test(two-tailed unpaired t test, t = -4.889, p = 0.000, n = 10 mice/group). (C) forced swimming test(two-tailed unpaired t test, t = -3.876, p = 0.001, n = 10 mice/group). (D-E) sucrose preference test(Mann-Whitney, p = 0.000; two-tailed unpaired t test, t = 1.422, p = 0.172, respectively, n = 10 mice/group). (F) representative images of c-Fos staining in PVT from Ctrl and CRS mice. Scale bar = 100 μm. (G) representative image of co-localization between FosB and CamkIIα in PVT. Scale bar = 100 μm. (H) statistic result of c-Fos + cells regarding PVT (two-tailed unpaired t test, t = 5.159, p = 0.007, n = 3 mice/group, each mice at least three brain slices). (I) FosB mRNA level in Ctrl and CRS mice(two-tailed unpaired t test, t = 3.294, p = 0.009, n = 6/5 mice for Ctrl/CRS group). (J) c-Fos mRNA expression in PVT(two-tailed unpaired t test, t = 3.733, p = 0.006, n = 5 mice/group). (K) statistic result of FosB + and CamkIIα + cells(two-tailed unpaired t test, t = 4.299, p = 0.005, n = 4 mice/group, each mice at least three brain slices). (L) the percentage of co-expression of FosB + and CamkIIα + cells. Data are expressed as mean ± SEM. ** p＜0.01, ***p＜0.001; n.s., not significant.

Article Snippet: In detail, c-Fos (1:1000, Novus, NBP2-50057), NPY (1:1000, CST, #11976), FosB (1:800, CST, #2251), CamkIIα (1:500, abcam, ab134041), CamkIIα (1:100, Santa Cruz, sc-13141), NeuN (1:1000, abcam, ab104224), GAD67 (1:500, abcam, ab26116), Vgat (1:500, Synaptic Systems, 131011).

Techniques: Activity Assay, Suspension, Two Tailed Test, MANN-WHITNEY, Staining, Expressing

Journal: Journal of Advanced Research

Article Title: Neural and molecular investigation into the paraventricular thalamus for chronic restraint stress induced depressive-like behaviors

doi: 10.1016/j.jare.2024.10.025

Figure Lengend Snippet: Activating PVT CamkIIα neurons improved anti-anxiety and anti-depressive level in naive mice, while inhibiting these neurons directly caused anxiety- and depressive-like behaviors. (A) Timeline of experimental design. (B) Illustration of virus delivery. (C) injection site of the virus. Scale bar = 100 μm. (D) tail suspension test(one-way ANOVA, F(2,26) = 3.142, p = 0.060, n = 9/10/10 mice for each group). (E) forced swimming test(one-way ANOVA, F(2,27) = 6.256, p = 0.006, n = 10 mice/group). (F-G) sucrose preference test(one-way ANOVA, F(2,27) = 4.376, p = 0.023; one-way ANOVA, F(2,27) = 0.620, p = 0.546, n = 10 mice/group). (H) representative images of c-Fos staining among three groups. Scale bar = 100 μm. (I) statistic result of c-Fos + cells in PVT(one-way ANOVA, F(2,7) = 17.398, p = 0.002, n = 3/4/3 mice for each group, each mice at least three brain slices). (J) the percentage of co-expression of c-Fos + and hM3D-CamkIIα + cells. (K) schematic timeline of the experimental procedure. (L) Illustration of virus injection. (M) representative image of viral injection site. Scale bar = 100 μm. (N) tail suspension test(one-way ANOVA, F(2,26) = 9.368, p = 0.001, n = 10/10/9 mice for each group). (O) forced swimming test(one-way ANOVA, F(2,26) = 5.561, p = 0.010, n = 10/10/9 mice for each group). (P-Q) sucrose preference test(one-way ANOVA, F(2,26) = 13.043, p = 0.000; one-way ANOVA, F(2,26) = 0.259, p = 0.774, n = 10/10/9 mice for each group). (R) Representative images of c-Fos staining among three groups. Scale bar = 100 μm. (S) statistic results of c-Fos + cells(one-way ANOVA, F(2,7) = 12.895, p = 0.004, n = 3/4/3 mice for each group, each mice at least three brain slices). (T) the percentage of co-expression of c-Fos + and hM4D-CamkIIα + cells. Data are expressed as mean ± SEM. *p＜0.05,** p＜0.01, ***p＜0.001; n.s., not significant.

Article Snippet: In detail, c-Fos (1:1000, Novus, NBP2-50057), NPY (1:1000, CST, #11976), FosB (1:800, CST, #2251), CamkIIα (1:500, abcam, ab134041), CamkIIα (1:100, Santa Cruz, sc-13141), NeuN (1:1000, abcam, ab104224), GAD67 (1:500, abcam, ab26116), Vgat (1:500, Synaptic Systems, 131011).

Techniques: Virus, Injection, Suspension, Staining, Expressing

Journal: Journal of Advanced Research

Article Title: Neural and molecular investigation into the paraventricular thalamus for chronic restraint stress induced depressive-like behaviors

doi: 10.1016/j.jare.2024.10.025

Figure Lengend Snippet: PVT CamkIIα neurons is essential for depressive-like behaviors. (A) schematic representation of experimental procedure. (B) illustration of virus injection. (C) representative images of Neun staining between Ctrl and lesion mice in PVT. Scale bar = 100 μm. (D-E) elevated plus maze(Mann-Whitney, p = 0.156; two-tailed unpaired t test, t = -0.972, p = 0.345, n = 9 mice/group). (F-H) open field test(two-tailed unpaired t test, t = -1.479, p = 0.159; Mann-Whitney, p = 0.402; two-tailed unpaired t test, t = 0.024, p = 0.981, n = 9 mice/group). (I) tail suspension test(two-tailed unpaired t test, t = -3.585, p = 0.002, n = 9 mice/group). (J) forced swimming test(two-tailed unpaired t test, t = -2.957, p = 0.009, n = 9 mice/group). (K-L) sucrose preference and total liquid intake(two-tailed unpaired t test, t = 6.462, p = 0.000; two-tailed unpaired t test, t = -2.024, p = 0.060, n = 9 mice/group). Data are expressed as mean ± SEM. ** p＜0.01, ***p＜0.001; n.s., not significant.

Article Snippet: In detail, c-Fos (1:1000, Novus, NBP2-50057), NPY (1:1000, CST, #11976), FosB (1:800, CST, #2251), CamkIIα (1:500, abcam, ab134041), CamkIIα (1:100, Santa Cruz, sc-13141), NeuN (1:1000, abcam, ab104224), GAD67 (1:500, abcam, ab26116), Vgat (1:500, Synaptic Systems, 131011).

Techniques: Virus, Injection, Staining, MANN-WHITNEY, Two Tailed Test, Suspension

Journal: Journal of Advanced Research

Article Title: Neural and molecular investigation into the paraventricular thalamus for chronic restraint stress induced depressive-like behaviors

doi: 10.1016/j.jare.2024.10.025

Figure Lengend Snippet: Activation of PVT CamkIIα neurons alleviate anxiety-like and depressive-like behaviors induced by CRS, whereas inhibition of these neurons increased the susceptibility to stress in mice. (A) timeline of experimental design. (B) representative image of virus injection site. Scale bar = 100 μm. (C-D) elevated plus maze(one-way ANOVA, F(2,26) = 2.990, p = 0.068; one-way ANOVA, F(2,26) = 8.591, p = 0.001, n = 10/10/9 mice for each group). (E-G) open field test(one-way ANOVA, F(2,26) = 5.508, p = 0.010; one-way ANOVA, F(2,26) = 5.706, p = 0.009; one-way ANOVA, F(2,26) = 2.850, p = 0.076, n = 10/10/9 mice for each group). (H) tail suspension test(one-way ANOVA, F(2,26) = 4.970, p = 0.015, n = 10/10/9 mice for each group). (I) forced swimming test (one-way ANOVA, F(2,26) = 11.576, p = 0.000, n = 10/10/9 mice for each group). (J-K) sucrose preference and total liquid consumption among three groups(one-way ANOVA, F(2,26) = 11.880, p = 0.000; one-way ANOVA, F(2,26) = 0.633, p = 0.539, n = 10/10/9 mice for each group). (L) schematic timeline of the experimental design. (M) representative image of viral injection. Scale bar = 100 μm. (N-O) elevated plus maze(two-tailed unpaired t test, t = 3.361, p = 0.003; two-tailed unpaired t test, t = -3.570, p = 0.002, n = 10 mice/group). (P-R) open field test(Mann-Whitney, p = 0.013; Mann-Whitney, p = 0.010; two-tailed unpaired t test, t = 0.660, p = 0.518, n = 10 mice/group). (S) tail suspension test(two-tailed unpaired t test, t = -2.450, p = 0.025, n = 10 mice/group). (T) forced swimming test(two-tailed unpaired t test, t = -5.078, p = 0.000, n = 10 mice/group). (U-V) sucrose preference test (Mann-Whitney, p = 0.000; two-tailed unpaired t test, t = -1.594, p = 0.128, n = 10 mice/group). Data are expressed as mean ± SEM. *p＜0.05,** p＜0.01, ***p＜0.001; n.s., not significant.

Article Snippet: In detail, c-Fos (1:1000, Novus, NBP2-50057), NPY (1:1000, CST, #11976), FosB (1:800, CST, #2251), CamkIIα (1:500, abcam, ab134041), CamkIIα (1:100, Santa Cruz, sc-13141), NeuN (1:1000, abcam, ab104224), GAD67 (1:500, abcam, ab26116), Vgat (1:500, Synaptic Systems, 131011).

Techniques: Activation Assay, Inhibition, Virus, Injection, Suspension, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Advanced Research

Article Title: Neural and molecular investigation into the paraventricular thalamus for chronic restraint stress induced depressive-like behaviors

doi: 10.1016/j.jare.2024.10.025

Figure Lengend Snippet: The expression of NPY were associated with activity of PVT CamkIIα neurons. (A) representative images of NPY staining between mCherry-CNO, hM3D-CNO and hM3D-saline mice. Scale bar = 100 μm. (B) statistical results (one-way ANOVA, F(2,8) = 14.656, p = 0.002, n = 4/3/4 mice for each group, each mice at least three brain slices). (C) representative images of NPY staining between mCherry-CNO, hM4D-CNO and hM4D-saline mice. Scale bar = 100 μm. (D) statistical results (one-way ANOVA, F(2,8) = 13.607, p = 0.003, n = 4/3/4 mice for each group, each mice at least three brain slices). (E) representative images of NPY staining between no stress-saline, SRS-saline and SRS-CNO mice. Scale bar = 100 μm. (F) statistical results (one-way ANOVA, F(2,6) = 11.024, p = 0.010, n = 3 mice/group, each mice at least three brain slices). (G) representative images of NPY staining between no stress-saline, CRS-saline and CRS-CNO mice. Scale bar = 100 μm. (H) statistical results (one-way ANOVA, F(2,6) = 5.355, p = 0.046, n = 3 mice/group, each mice at least three brain slices). Data are expressed as mean ± SEM. *p＜0.05,** p＜0.01, n.s., not significant.

Article Snippet: In detail, c-Fos (1:1000, Novus, NBP2-50057), NPY (1:1000, CST, #11976), FosB (1:800, CST, #2251), CamkIIα (1:500, abcam, ab134041), CamkIIα (1:100, Santa Cruz, sc-13141), NeuN (1:1000, abcam, ab104224), GAD67 (1:500, abcam, ab26116), Vgat (1:500, Synaptic Systems, 131011).

Techniques: Expressing, Activity Assay, Staining, Saline

Journal: bioRxiv

Article Title: NR2F2 Reactivation in Early-life Adipocyte Stem-like Cells Rescues Adipocyte Mitochondrial Oxidation

doi: 10.1101/2024.09.09.611047

Figure Lengend Snippet: (A) mRNA expression of Nr2f2, Axin2, and Sptlc2 in ASCs ± Wnt agonist 1(48h) quantified through qPCR (n=3 wells per group). (B) Model showing effects of excess n6-FA exposure on ASCs and how that affects adipocyte metabolism. ASCs exposed to n6-FA ratio have a diminished WNT mediated activation of CTNNb1 gene signature, leading to poor induction of NR2F2 and lower expression of beige regulators PPARγ, PGC1α, Cidea, and Prdm16. These ASCs differentiate, give rise to mature adipocytes with diminished electron transport chain and oxidative phosphorylation protein components, blunted nutrient oxidation, while retaining an enhanced capacity for triacylglyceride accumulation. Data are expressed as mean ± SEM, statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA.

Article Snippet: Lysates were run on a ProteinSimple JESS instrument for PPARγ (CST-2435), PGC1α (CST-2178), C/EBPα (CST-2295), UCP1 (CST-14670), CPT1A (Protein Tech-15184-1-AP), CPT1C (Protein Tech-12969-1-AP), FABP4 (CST-2120), NR2F2 (CST-6434), SOD2 (CST-13141), TOM20 (CST-42406), and PLIN1 (CST-9349) antibodies.

Techniques: Expressing, Activation Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: NR2F2 Reactivation in Early-life Adipocyte Stem-like Cells Rescues Adipocyte Mitochondrial Oxidation

doi: 10.1101/2024.09.09.611047

Figure Lengend Snippet: (A) Diagram of experimental design. (B) Body composition of control and high n6-FA exposed pups at Postnatal day 12 (PND12). Each data point is the mean mass, lean body mass, fat mass, ratio of fat to lean mass, or fat mass percentage of one litter of pups at PND12 (n=13-15 litters). (C) Indirect calorimetry of control and high n6-exposed litters. Dams were removed for the 2h pre- and 3h post-nursing periods to measure the Respiratory Exchange Ratio (VCO2/VO2: Scale 0.5-1.0) of pups in a litter. Data are presented as means ± SEM (n=7-9). (D) Fatty acid oxidation (FAO) calculated using the equation (1.70 × VO 2 – 1.69 × VCO 2 ) (n=7-9). (E) Pups were administered 13 C 16 -palmitate (100 mg/kg body weight) and the litters placed into cages in an Indirect Calorimeter cabinet held at 25°C to quantify 13 CO 2 stable isotope gas exchange as a measure of whole-body FAO (n=3 per group). (F) H&E staining of control and n6-FA exposed pup SAT (inguinal fat) and quantification of cellularity (n=7-8). Scale bar is 100 μm. (G) Quantification of SAT triglyceride (TAG) levels (n=5). (H) Immunofluorescence staining of inguinal adipose tissue with anti-PLIN1 antibody (green) and DAPI (blue) (n=5 neonatal inguinal SAT sections from independent litters per control/n6-FA group). The scale bar is 100 μm. Data are expressed as mean ± SEM, statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t-test.

Article Snippet: Lysates were run on a ProteinSimple JESS instrument for PPARγ (CST-2435), PGC1α (CST-2178), C/EBPα (CST-2295), UCP1 (CST-14670), CPT1A (Protein Tech-15184-1-AP), CPT1C (Protein Tech-12969-1-AP), FABP4 (CST-2120), NR2F2 (CST-6434), SOD2 (CST-13141), TOM20 (CST-42406), and PLIN1 (CST-9349) antibodies.

Techniques: Control, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: NR2F2 Reactivation in Early-life Adipocyte Stem-like Cells Rescues Adipocyte Mitochondrial Oxidation

doi: 10.1101/2024.09.09.611047

Figure Lengend Snippet: (A) Immunofluorescence staining of inguinal SAT from PND12 mice with anti-UCP1 (red) and anti-PLIN1 (green) antibodies. Nuclei (blue) were stained with DAPI. Morphologically defined white and beige regions were quantified separately for UCP1 staining, which was significantly decreased in n6-FA group (n=8-11 neonatal SAT section from independent litters per control/n6 group). (B, C) Analysis of UCP1, SOD2, and TOM20 in inguinal fat pads through Western blotting. CypA was used as the reference protein to normalize loading (n=3-6 neonatal SAT tissue from independent litters per control/n6-FA group). (D, E) Levels of key adipocyte proteins and mRNA were measured through Western blotting and qPCR in mature adipocytes isolated from inguinal fat from control and n6-FA pups. (n=3-4 pooled adipocytes from neonatal SAT tissue from independent litters per control/n6-FA group). Data are expressed as mean ± SEM, statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t-test.

Article Snippet: Lysates were run on a ProteinSimple JESS instrument for PPARγ (CST-2435), PGC1α (CST-2178), C/EBPα (CST-2295), UCP1 (CST-14670), CPT1A (Protein Tech-15184-1-AP), CPT1C (Protein Tech-12969-1-AP), FABP4 (CST-2120), NR2F2 (CST-6434), SOD2 (CST-13141), TOM20 (CST-42406), and PLIN1 (CST-9349) antibodies.

Techniques: Immunofluorescence, Staining, Control, Western Blot, Isolation

Journal: bioRxiv

Article Title: NR2F2 Reactivation in Early-life Adipocyte Stem-like Cells Rescues Adipocyte Mitochondrial Oxidation

doi: 10.1101/2024.09.09.611047

Figure Lengend Snippet: (A) Immunofluorescence staining of inguinal SAT from PND12 mice with anti-UCP1 (red) and anti-PLIN1 (green) antibodies. Nuclei (blue) were stained with DAPI. Morphologically defined white and beige regions were quantified separately for UCP1 staining, which was significantly decreased in n6-FA group (n=8-11 neonatal SAT section from independent litters per control/n6 group). (B, C) Analysis of UCP1, SOD2, and TOM20 in inguinal fat pads through Western blotting. CypA was used as the reference protein to normalize loading (n=3-6 neonatal SAT tissue from independent litters per control/n6-FA group). (D, E) Levels of key adipocyte proteins and mRNA were measured through Western blotting and qPCR in mature adipocytes isolated from inguinal fat from control and n6-FA pups. (n=3-4 pooled adipocytes from neonatal SAT tissue from independent litters per control/n6-FA group). Data are expressed as mean ± SEM, statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t-test.

Article Snippet: Lysates were run on a ProteinSimple JESS instrument for PPARγ (CST-2435), PGC1α (CST-2178), C/EBPα (CST-2295), UCP1 (CST-14670), CPT1A (Protein Tech-15184-1-AP), CPT1C (Protein Tech-12969-1-AP), FABP4 (CST-2120), NR2F2 (CST-6434), SOD2 (CST-13141), TOM20 (CST-42406), and PLIN1 (CST-9349) antibodies.

Techniques: Immunofluorescence, Staining, Control, Western Blot, Isolation