Journal: BMC Biology

Article Title: 20-Hydroxyecdysone counteracts insulin to promote programmed cell death by modifying phosphoglycerate kinase 1

doi: 10.1186/s12915-023-01621-2

Figure Lengend Snippet: PGK1 knockdown at the wandering stage delays metamorphosis. A Morphology, HE staining, and caspase-3 location in the fat body after dsGFP and dsPGK1 knockdown. PGK1 knockdown (500 ng/larva administered in the sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 60 h. dsGFP was used as a negative control. Rabbit polyclonal antibodies against caspase-3 were used as the primary antibody. Green fluorescence indicates caspase-3 presence. Nuclei were stained with DAPI (blue). A’ Quantification of fluorescent caspase-3 in A . ** p < 0.01, n = 3. B TEM analysis of the fat body from A . LD indicates lipid droplets. Red arrows indicate autolysosomes or autophagosomes. Blue arrows represent the apoptotic nuclei. B’ Quantification of autophagosomes, autolysosomes, and apoptotic nucleus in B . * p < 0.05, n = 3. C and C’ The levels of ATG8-II and cleaved-caspase-3 in the fat body based on A . Western blot detection using anti-ATG8 and anti-caspase-3 antibodies. * p < 0.05, n = 3. D and D’ PGK1 acetylation levels in dsGFP and dsPGK1 fat body from A . PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with anti-PGK1 monoclonal antibody. * p < 0.05, n = 3. E Interference efficiency analysis of PGK1 at protein levels from the input of D . * p < 0.05, n = 3. F The Vmax of 3-PG production was determined from A in vitro. PGK1 proteins from the fat body were purified by CNBr-activated Sepharose 4B with the anti-PGK1 monoclonal antibody. n = 3. G and H Concentrations of glucose ( G ) and lactate ( H ) in H. armigera hemolymph from A . n = 3. I Phenotypes after PGK1 knockdown as A (500 ng/larva administered in sixth-instar at 72 h, three times over 24 h intervals). Images were obtained after the first RNAi exposure for 80 h. I’ Percentages of phenotypes in I based on three repeats (thirty larvae for each repeat). * p < 0.05, ** p < 0.01, n = 3. J The pupation time from 6th-6 h to pupa 0 d after RNAi exposure in I . * p < 0.05, n = 3. K The average body weight of pupa after RNAi exposure in I . n = 3. Bars indicate means ± SD for three independent experiments with five larvae per replicate. Statistical analyses were performed using two-tailed Student’s t tests (*: p < 0.05 and **: p < 0.01)

Article Snippet: Antibodies were commercially purchased including mouse monoclonal antibody anti-PGK1 (Santa Cruz Biotechnology, Cat. sc-130335; RRID: AB_627677), mouse monoclonal antibody anti-phosphate-tyrosine (anti-pY; Santa Cruz Biotechnology, Cat. sc-7020; RRID: AB_628123), rabbit polyclonal antibodies anti-acetylated lysine (anti-Ac; Cell Signaling Technology, Cat. 9441), rabbit monoclonal antibody anti-β-actin (ABclonal, Cat. AC026), mouse anti-GFP-Tag mAb (ABclonal, Cat. AE012), mouse anti-His-Tag mAb (ABclonal, Cat. AE003), mouse control IgG (ABclonal, Cat. AC011), and alkaline phosphatase-labeled goat anti-rabbit or horse anti-mouse IgG secondary antibodies (ZSGB-BIO, Cat. ZB-2308; ZB-2310).

Techniques: Staining, Negative Control, Fluorescence, Western Blot, Purification, In Vitro, Two Tailed Test

Journal: Cell reports

Article Title: Downregulation of hepatic ceruloplasmin ameliorates NAFLD via SCO1-AMPK-LKB1 complex

doi: 10.1016/j.celrep.2022.111498

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-acetylated-Lysine antibody, WB, dil: 1/1,000 , Cell Signaling Technology , Cat#9441; RRID: AB_331805.

Techniques: Magnetic Beads, shRNA, Recombinant, Protease Inhibitor, Lysis, Modification, Transfection, BIA-KA, Mutagenesis, Purification, Reporter Assay, TUNEL Assay, RNA Sequencing Assay, Transgenic Assay, Plasmid Preparation, Software

Journal: iScience

Article Title: Histone malonylation is regulated by SIRT5 and KAT2A

doi: 10.1016/j.isci.2023.106193

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-acetyl-lysine , Cell Signaling Technology , Cat. #9814, RRID: AB_10544700.

Techniques: Recombinant, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Reduced glycolysis links resting zone chondrocyte proliferation in the growth plate

doi: 10.1101/2023.01.18.524550

Figure Lengend Snippet: (A) Reduced cytosolic citrate and acetyl-CoA (Ac-CoA) levels in Mir140UGCG mice. Ac-CoA and citrate levels were determined in primary rib chondrocytes from 10-day-old mice. (B) Immunofluorescence of indicated acetylated histones in the spheno-occipital growth plate. Dotted boxes indicate the resting zone. Insets are magnified views of the resting zone. MIr1430UGCG mice show reduced histone acetylation. (C) Immunoblot analysis for H3K27 acetylation. (D) Immunofluorescence assessment of H3K27 acetylation in the tibial growth plate of control and Ldha cKO mice. Insets are magnified views of dotted boxes. (E) Quantification of H3K27 acetylation (H3K27Ac) in D . H3K27Ac-positive cells were counted in the resting zone and proliferating zone and normalized to the total number of cells per area. Ldha cKO show reduced numbers of chondrocytes positive for H3K27Ac. (F) Immunoblot quantification of acetylation of indicated histones in primary rib chondrocytes. Ldha cKO shows reduced histone acetylation.

Article Snippet: Immunofluorescence was performed according to the protocol described in the data sheets of the anti-acetylated histone 3 lysine 27 (H3K27Ac) antibody (# 8173, Cell Signaling Technology) and anti-acetylate histone 3 lysine 9 (H3K9Ac) antibody (#9649, Cell Signaling Technology).

Techniques: Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: Reduced glycolysis links resting zone chondrocyte proliferation in the growth plate

doi: 10.1101/2023.01.18.524550

Figure Lengend Snippet: (A) Acly cKO mice show shortening and bowing of the tibia ( Top) . Mice are perinatally lethal. H/E-stained sections of the tibia at P 0.5. The bottom panels show magnified views of proliferating chondrocytes in the boxed areas. The flat morphology of the columnar chondrocytes is lost in cKO mice. (B) Expansion of the resting zone chondrocytes in the spheno-occipital growth plate in E18.5 Acly cKO mice. Left , H/E-stained ( Top ) and EdU-stained ( Bottom ) sections. Boxed areas indicate the resting zone. Right , The number of resting chondrocytes is increased in the Acly cKO growth plate. (C) Reduced histone 3 acetylation in primary rib chondrocytes from E18.5 Acly cKO mouse embryos. Whole-cell lysates from an overnight culture of primary rib chondrocytes were subjected to immunoblot analysis. Acly cKO chondrocytes show reduced acetylation of H3K27 and H3K9. (D). Differential phosphorylation of AMPK between Acly cKO and Ldh compound cKO chondrocytes. The level of p-AMPK is unchanged in Acly cKO, whereas it is upregulated in Ldh compound cKO cells, suggesting ATP deficiency in Ldh compound cKO, but not in Acly cKO chondrocytes.

Article Snippet: Immunofluorescence was performed according to the protocol described in the data sheets of the anti-acetylated histone 3 lysine 27 (H3K27Ac) antibody (# 8173, Cell Signaling Technology) and anti-acetylate histone 3 lysine 9 (H3K9Ac) antibody (#9649, Cell Signaling Technology).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Reduced glycolysis links resting zone chondrocyte proliferation in the growth plate

doi: 10.1101/2023.01.18.524550

Figure Lengend Snippet: (A) Acly cKO mice show shortening and bowing of the tibia ( Top) . Mice are perinatally lethal. H/E-stained sections of the tibia at P 0.5. The bottom panels show magnified views of proliferating chondrocytes in the boxed areas. The flat morphology of the columnar chondrocytes is lost in cKO mice. (B) Expansion of the resting zone chondrocytes in the spheno-occipital growth plate in E18.5 Acly cKO mice. Left , H/E-stained ( Top ) and EdU-stained ( Bottom ) sections. Boxed areas indicate the resting zone. Right , The number of resting chondrocytes is increased in the Acly cKO growth plate. (C) Reduced histone 3 acetylation in primary rib chondrocytes from E18.5 Acly cKO mouse embryos. Whole-cell lysates from an overnight culture of primary rib chondrocytes were subjected to immunoblot analysis. Acly cKO chondrocytes show reduced acetylation of H3K27 and H3K9. (D). Differential phosphorylation of AMPK between Acly cKO and Ldh compound cKO chondrocytes. The level of p-AMPK is unchanged in Acly cKO, whereas it is upregulated in Ldh compound cKO cells, suggesting ATP deficiency in Ldh compound cKO, but not in Acly cKO chondrocytes.

Article Snippet: Immunofluorescence was performed according to the protocol described in the data sheets of the anti-acetylated histone 3 lysine 27 (H3K27Ac) antibody (# 8173, Cell Signaling Technology) and anti-acetylate histone 3 lysine 9 (H3K9Ac) antibody (#9649, Cell Signaling Technology).

Techniques: Staining, Western Blot