Journal: Scientific Reports

Article Title: Ca +2 and Nε-lysine acetylation regulate the CALR-ATG9A interaction in the lumen of the endoplasmic reticulum

doi: 10.1038/s41598-024-76854-4

Figure Lengend Snippet: Ca +2 -binding sites on CALR are required to maintain normal binding with ATG9A. ( A ) Structural model of CALR (AF-P27797-F1) showing Ca +2 binding sites in orange. ( B-G ) Co-immunoprecipitation of ATG9A and CALR in HEK293 cells overexpressing tGFP tagged ATG9A and WT or mutant DDK tagged CALR. CALR mutations targeted the high-affinity/low-capacity Ca +2 -binding site (D328A; see arrow in panel A ) and the low-affinity/high-capacity Ca +2 -binding α-helix (Δ338–408; shown in orange in panel A ). Representative Western blots and accompanying quantification, normalized to the IP-ed protein, are shown. The upper band of the ATG9A blots corresponds to the mature, glycosylated form whereas the lower band corresponds to the immature, unglycosylated form that is found in the ER. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 via Student’s t test. ( H ) Absorbance and molar ellipticity of CALR incubated with 0 mM, 5 mM, or 20 mM CaCl 2 as measured with circular dichroism. ( I ) Flow cytometry analysis for elevated reticulophagy flux in CRISPR-modified HEK293 EATR cells ( left panel ). CALR CR cells have targeted mutations to both CALR Ca +2 binding sites and the WT cells are no-guide controls. Media was changed to fresh complete DMEM for the fed condition and to EBSS for the staved condition 2 h prior to flux analysis. Western blot ( right panel ) shows the migration profile of CALR CR . Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 via Student’s t test ( B - G ) or ordinary one-way ANOVA with Sidak’s multiple comparison test ( I ). Each point represents an individual biological sample.

Article Snippet: The following primary antibodies were used in this study: DDK (Origene, TA100011, 1:1000), ITPR3 (abcam, ab264283, 1:2000), tGFP (Origene, TA150071, 1:1000), and HA (Cell Signaling Technology, C29F4, 1:1000).

Techniques: Binding Assay, Immunoprecipitation, Mutagenesis, Western Blot, Incubation, Circular Dichroism, Flow Cytometry, CRISPR, Modification, Migration, Comparison

Journal: Scientific Reports

Article Title: Ca +2 and Nε-lysine acetylation regulate the CALR-ATG9A interaction in the lumen of the endoplasmic reticulum

doi: 10.1038/s41598-024-76854-4

Figure Lengend Snippet: ATG9A endoluminal acetylation mediates interaction with CALR and subcellular localization. ( A-B ) Co-immunoprecipitation of CALR and ATG9A in HEK293 cells overexpressing DDK tagged CALR and WT or mutant tGFP tagged ATG9A. Representative Western blots and accompanying quantification, normalized to the interaction between CALR and WT ATG9A, are shown. Each point represents an individual biological sample. ( C-D ) Representative images and accompanying Pearson’s colocalization analysis for HEK293 cells transiently expressing ATG9A-tGFP and ERmCherry. Each point represents a cell (WT n = 35; ΔK359 + ΔK363 n = 39). Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 via Student’s t test or ordinary one-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: The following primary antibodies were used in this study: DDK (Origene, TA100011, 1:1000), ITPR3 (abcam, ab264283, 1:2000), tGFP (Origene, TA150071, 1:1000), and HA (Cell Signaling Technology, C29F4, 1:1000).

Techniques: Immunoprecipitation, Mutagenesis, Western Blot, Expressing, Comparison

Journal: JCI Insight

Article Title: Brd4 modulates diet-induced obesity via PPAR γ -dependent Gdf3 expression in adipose tissue macrophages

doi: 10.1172/jci.insight.143379

Figure Lengend Snippet: ( A ) PCA of RNA-Seq data derived from CD11b + ATMs of WT and Brd4 -CKO mice fed a HFD for 20 weeks. ( B ) Volcano plot of mRNA-Seq analysis in ATMs as indicated in A . Brown dots represent genes increased in ATMs of Brd4 -CKO mice vs. WT mice (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value). Blue dots represent genes decreased in ATMs of Brd4 -CKO mice vs. WT mice (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value). Gray dots represent genes without significantly altered expression. Clusters of significantly altered genes (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value) were identified using gene ontology terms ( C ) and KEGG pathways ( D ). ( E ) Heat map of the relative expression levels (scaled Z -score) of cytokine-cytokine receptor interaction-related genes clustered in ( D ). ( F ) mRNA levels of Gdf3 in CD11b + ATMs isolated from WT or Brd4 -CKO mice fed a HFD for 20 weeks. ( G ) Left panel: Gdf3 or F4/80 IHC staining of eWAT of WT or Brd4-CKO mice fed a HFD for 20 weeks. Right panel: statistical analysis of Gdf3-positive or F4/80-positive area percentage, the ratio of Gdf3-positive cells in F4/80-positive cells in eWAT of WT and Brd4 -CKO mice fed a HFD. Data are mean and SD and are determined by an unpaired 2-tailed Student’s t test. n = 3 mice. ** P < 0.01. Brd4 -CKO, myeloid lineage-specific Brd4 knockout; HFD, high-fat diet–induced; eWAT, epididymal WAT; ATMs, adipose tissue macrophages; PCA, principal component analysis.

Article Snippet: Four-μm continuous sections were prepared for immunostaining with Gdf3 antibody (AF958, R&D Systems) or F4/80 antibody (BM4008, Origene).

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Isolation, Immunohistochemistry, Knock-Out

Journal: International Journal of Biological Sciences

Article Title: NLRP3 Regulates Mandibular Healing through Interaction with UCHL5 in MSCs

doi: 10.7150/ijbs.78174

Figure Lengend Snippet: NLRP3 inflammasome activation in MSC-mediated mandibular healing. Two-month-old WT or Prx1-Cre/ROSA nTnG mice were used, as specified in the figure legends. Mice received mandibular osteotomy surgery and were sacrificed at various time points during healing, N=4. (A) Representative images of ALP-stained paraffin sections show mandible defects of WT mice. ALP-positive area relative to tissue area (%) was determined. (B) Representative images of TRAP-stained paraffin sections show defect areas of WT mice. The surface of osteoclasts relative to the bone surface (%) was determined. (C) Representative images of unstained frozen sections under fluorescence microscopy and H&E-stained adjacent sections of mandible defects at POD 14 of Prx1-Cre/ROSA nTnG mice. Images show Prx1-Cre + cells (GFP, green) and Cre - cells (tdTomato, red) in defect areas. Prx1-Cre + or Cre - cell number relative to all cell number (%) was assessed. (D) Representative IHC images for NLRP3 in defect areas at various time points. The percentage of NLRP3-positive area was calculated. (E) Paraffin sections of WT mice at POD 7 were subjected to double immunofluorescence staining with anti-Prx1 and anti-NLRP3. Representative images of defect areas are shown. The proportion of NLPR3 + Prx1 + or NLPR3 + Prx1 - area to NLRP3 + area (%) were determined. (F-H) M-MSCs from WT mice were cultured and stimulated with or without 10 μg/ml LPS ± 5 mM ATP during culture, as indicated in the figures. (F) Western blot of cell lysates and supernatants from M-MSCs cultures. (G) The relative gene expression levels of Col1α and OCN were determined by qPCR. (H) M-MSCs were cultured in osteoblast-inducing medium for 7 days. Culture plates were stained for ALP, and ALP + areas were measured. Scale bars, 200 μm (A, B and D) and 100 μm (C and E). All error bars represent SEM. Two-tailed unpaired Student's t-test was performed. * P < 0.05 vs. POD 0 or in the indicated groups.

Article Snippet: The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H 2 O 2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against NLRP3 (Abcam, Cat#ab214185), Prx1 (Origene, Cat#TA803116), USP1 (CST, Cat#D37B4) or UCHL5 (Abcam, Cat#ab133508) at 4 °C.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, Double Immunofluorescence Staining, Cell Culture, Western Blot, Expressing, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: NLRP3 Regulates Mandibular Healing through Interaction with UCHL5 in MSCs

doi: 10.7150/ijbs.78174

Figure Lengend Snippet: NLRP3 deficiency promotes osteoblast differentiation and mandibular healing. Two-month-old WT, NLRP3 KO , or Prx1-Cre/ROSA nTnG mice were used. (A) Schematic of experimental design. (B-D) Frozen sections of defect areas from Prx1-cre/Rosa nTnG mice received locol injection of MCC950 or PBS were used, N=4. (B) Representative images of unstained frozen sections under fluorescence microscopy and H&E-stained adjacent sections of mandible defects at POD 14 of Prx1-Cre/ROSA nTnG mice. Prx1-Cre + area (%) was assessed. The number of Prx1-Cre + cells along the bone surface relative to the number of total Prx1-Cre + cells (%) was determined. (C) Representative images of ALP-stained frozen sections show mandible defects. ALP-positive area relative to tissue area (%) was determined. (D) Representative reconstructed sections of mandibles (upper panels) and defects (lower panels) and morphometric data of bone volume (mm 3 ) relative to tissue volume (%). (E) Primary cultures of mandibular bone marrow cells from WT and NLRP3 KO mice were stained with methylene blue to show total CFU-F colonies, stained cytochemically for ALP to detect CFU-ALP + colonies, or stained with Alizarin Red to show mineralized nodule formation. The number of CFU-F colonies, CFU-ALP + colonies and nodules per well (#/well) were counted. (F-I) Two-month-old WT and NLRP3 KO mice received mandibular osteotomy surgery and were sacrificed at various time points during healing, N=6. (F) Representative reconstructed sections of mandibles (upper panels) and defects (lower panels). (G) Morphometric data of bone volume (mm 3 ) relative to tissue volume (%). (H) Representative images of ALP-stained paraffin sections show mandible defects. (I) ALP-positive area relative to tissue area (%) was determined. Scale bars, 200 μm. All error bars represent SEM. Two-tailed unpaired Student's t-test was performed. * P < 0.05 in the indicated groups.

Article Snippet: The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H 2 O 2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against NLRP3 (Abcam, Cat#ab214185), Prx1 (Origene, Cat#TA803116), USP1 (CST, Cat#D37B4) or UCHL5 (Abcam, Cat#ab133508) at 4 °C.

Techniques: Injection, Fluorescence, Microscopy, Staining, Two Tailed Test