prodh  (Proteintech)

Journal: Advanced Science

Article Title: Connexin43 Deficiency Leads to Ventricular Arrhythmias by Reprogramming Proline Metabolism

doi: 10.1002/advs.202516090

Figure Lengend Snippet: Downregulation of the Cx43‐interacting protein SNAT2 leads to disorders of proline metabolism and disturbances in redox balance of Cx43‐KO iPSC‐CMs. (A) Bar graph to compare the mRNA expression of the proline transporters ( SLC36A1 , SLC36A2 , SLC38A1 , and SLC38A2 ) between WT and Cx43‐KO iPSC‐CMs. n = 4 independently biological repeats. (B–D) Western blot analysis of the protein expression of SNAT2 (sodium‐coupled neutral amino acid transporter) and PRODH (proline dehydrogenase) in WT and Cx43‐KO iPSC‐CMs. GAPDH is used as the loading control. n = 3 independently biological repeats. (E) Co‐immunoprecipitation (co‐IP) assay showing that SNAT2 was detected in anti‐Cx43 immunoprecipitates in WT iPSC‐CMs. (F) Molecular docking simulation using AutoDockTools and GRAMM. Cx43 and SNAT2 are represented as slate and cyan cartoon models, respectively. (G) Measurement of proline contents through a proline assay kit in Cx43‐KO iPSC‐CMs transfected with green fluorescent protein (GFP) only (negative control, NC) (KO + NC) or SNAT2 protein labeled with GFP (SNAT2‐OE) (KO + SNAT2‐OE). OE, overexpressing. n = 5 independently biological repeats. (H) Bar graph to compare the mitochondrial ROS level among WT, KO + NC, and KO + SNAT2‐OE iPSC‐CMs. n = 4 independently biological repeats. (I) Bar graph to compare the GSH/GSSG among WT, KO + NC, and KO + SNAT2‐OE iPSC‐CMs. n = 3 independently biological repeats. (J) Bar graph to compare the proline content among WT iPSC‐CMs transfected with scrambled siRNA (WT + NC), WT iPSC‐CMs transfected with SNAT2 small interfering RNA (siRNA) (WT + SNAT2‐KD), and KO iPSC‐CMs. KD, knockdown. n = 5 independently biological repeats. (K–N) Bar graphs to compare the calcium amplitude, peak calcium, maximum rising rate, and maximum decay rate among WT + NC1, WT + SNAT2‐KD, KO + NC2, and KO + SNAT2‐OE iPSC‐CMs. n = 21–24 cells. (O) The diagram depicting the traces of OCR on WT and Cx43‐KO iPSC‐CMs treated with PBS (vehicle) or proline (100 n m , 24 h) after sequentially administration of 1.5 µ m oligomycin, 4 µ m FCCP, and 1 µ m antimycin A, respectively. (P–S) Bar graphs to compare a series of fundamental parameters of mitochondrial function among different groups in Panel O, including basal OCR, spare respiratory capacity, maximal respiration, and ATP production. n = 2–4 independently biological repeats. (T) Measurement of mitochondrial ROS levels in WT and Cx43‐KO iPSC‐CMs with or without ISO (100 n m , 2 h) stimulation and/or proline (100 n m , 24 h) supplementation. n = 4 independently biological repeats. (A–D,G–T) “KO” in the figure panels refers to combined data from KO‐1 and KO‐2 analyzed in parallel. (A,C,D,G) p values were calculated using unpaired two‐tailed Student's t ‐test, (H) Brown–Forsythe ANOVA test/Welch ANOVA test followed by Dunnett T3 multiple comparisons test, (I) one‐way ANOVA followed by Dunnett's multiple comparisons test, (J) one‐way ANOVA followed by Dunnett's multiple comparisons test, (K,M,N) Kruskal–Wallis test followed by Dunn's multiple comparisons test, (L,T) one‐way ANOVA followed by Tukey's multiple comparisons test, and (P–S) two‐way ANOVA followed by Tukey's multiple comparisons test. Data were shown as mean ± SEM.

Article Snippet: Western blot was performed using standard protocol with the following antibodies: Cx43 (Abcam, ab11370, 1:1000), DRP1 (Proteintech, 12957‐1‐AP, 1:500), phospho‐DRP1 (Ser616) (Cell signaling, 3455S, 1:1000), PRODH (Proteintech, 22980‐1‐AP, 1:500), SNAT2 (Santa Cruz Biotechnology, sc‐514037, 1:500), Na + /K + ‐ATPase (Abcam, ab7671, 1:1000), Na v 1.5 (Alomone labs, ASC005, 1:500), Ca v 1.2 (Abcam, ab84814, 1:1000), NCX1 (Proteintech, 55075‐1‐AP, 1:1000), SERCA2a (Santa Cruz Biotechnology, sc‐53010, 1:200), and GAPDH (Abmart, M200006, 1:5000).

Techniques: Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Transfection, Negative Control, Labeling, Small Interfering RNA, Knockdown, Two Tailed Test

Journal: Animal Nutrition

Article Title: Increased proline intake during gestation alleviates obesity-related impaired fetal development and placental function in gilts

doi: 10.1016/j.aninu.2024.10.007

Figure Lengend Snippet: Effects of body condition and dietary Pro level during gestation on placental mitochondrial complexes of gilts. (A) Immunoblots of NDUFB8 (CI), SDHA (CII), UQCRC1 (CIII), COXIV (CIV), and ATPB (CV) in the placentae of gilts. (B) Quantification of NDUFB8 (CI). (C) Quantification of SDHA (CII). (D) Quantification of UQCRC1 (CIII). (E) Quantification of COXIV (CIV). (F) Quantification of ATPB (CV). Statistical significance was determined by 2 × 3 factorial treatment arrangement, factor 1 = body conditions, factor 2 = dietary Pro levels. Nor = gilts with an average body mass index of 62.7 kg/m 2 and an average backfat thickness of 12.9 mm, Obe = gilts with an average body mass index of 67.6 kg/m 2 and an average backfat thickness of 14.4 mm, L-Pro = low Pro level (0.89%) diet, M-Pro = medium Pro level (1.39%) diet, H-Pro = high Pro level (1.89%) diet. Pro = proline; NDUFB8 (CI) = NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8; SDHA (CII) = succinate dehydrogenase complex flavoprotein subunit A; UQCRC1 (CIII) = ubiquinol-cytochrome c reductase core protein 1; COXIV (CIV) = cytochrome c oxidase IV subunit; ATPB (CV) = ATPase beta chain. n = 6 placentae from 6 litters per group. Mean values with ∗, #, and NS indicate significant (∗ P (body condition) < 0.05, ∗∗ P (body condition) < 0.01, ∗∗∗ P (body condition) < 0.001), trend (0.05 ≤ P (body condition) < 0.10), and no statistically difference ( P (body condition) ≥ 0.10) of significant body condition factor under the same diet, respectively; Mean values sharing no common letter (a to c) indicate a significant effect ( P (Pro) < 0.05) of dietary Pro levels factor.

Article Snippet: The levels of adenosine triphosphate (ATP; S0026, Beyotime, China), nicotinamide adenine dinucleotide reduced (NADH; S0175, Beyotime, China), nicotinamide adenine dinucleotide (NAD; S0175, Beyotime, China), malondialdehyde (MDA; S0131, Beyotime, China), Pro dehydrogenase (PRODH; BC4165, Solarbio, China), sirtuin 1 (Sirt1; MEIMIAN, China), and superoxide dismutase 2 (SOD2; A001-2-2, Nanjing Jiancheng Bioengineering Institute, China) were measured using commercial kits (Beyotime, China).

Techniques: Western Blot