Journal: Cell reports

Article Title: Quantitative trait loci mapping provides insights into the genetic regulation of dendritic cell numbers in mouse tissues

doi: 10.1016/j.celrep.2024.114296

Figure Lengend Snippet: (A) Manhattan plot showing all splice variant SNPs across the 49 phenotypes. For each splice variant, the best p value observed among all assessed traits is plotted on a –log10 scale (y axis), according to its genomic coordinates (x axis). Gramd4 splice variant chosen for validation is highlighted in red. (B) Variation in frequency of BM pre-cDCs in C57BL/6J (gray; n = 3), 129 (pink; n = 3), NOD (blue; n = 3), NZO (cyan; n = 3), A/J (yellow; n = 3), WSB (purple; n = 3), DO (black; n = 170), and CC-RI (open; n = 47) mice. (C) A QTL driving the frequency of BM pre-cDCs found within chromosome 15 (chr15:86,581,607, LOD = 10.7) that appears to be driven by an A/J and NZO founder effect. (D) Gramd4 gene structure and schematic representation of alternative splicing (UCSC Genome Browser). SNP localization is shown in red. (E) Western blot on total splenocytes from Gramd4 w/w and Gramd4 sp/sp mice showing alternative splicing. (F) Schematic representation of mixed BM chimera experiment. (G) Representative flow cytometry plot for mixed BM experiment. (H) Frequencies of DC progenitors and subsets in mixed BM chimera mice due to differential expression of Gramd4 SNP variant in BM, spleen, and inguinal LN. Chimerism is expressed as the ratio between the number of CD45.2 and CD45.1/CD45.2 cells for each cell population. Representative of 2 independent experiments; each dot represents one mouse, n = 6 per group, Gramd4 w/w (red) and Gramd4 sp/sp (blue), and horizontal lines represent means. (I) Schematic representation of the experimental setup in (J) and (K). (J) Representative flow cytometry plot for OT-II CD4 + T cell activation (CTV dilution after immunization with OVA 328–339 peptide in alum) in popliteal LN of Gramd4 w/w and Gramd4 sp/sp recipient mice. Graph shows the absolute numbers of OT-II cells in popliteal lymph nodes and the x axis the number of divisions after immunization. (K) CD69 and CD43 expression of divided OT-II T cells in popliteal LN of Gramd4 w/w and Gramd4 sp/sp recipient mice. Each dot represents one mouse, n = 6 per group, Gramd4 w/w (orange) and Gramd4 sp/sp (green), and horizontal lines represent means (J and K). Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. iLN (inguinal LN).

Article Snippet: After being blocked, the western blot membrane was subsequently incubated with anti-Gramd4 antibody (Clone C-8, Santa-Cruz).

Techniques: Variant Assay, Biomarker Discovery, Alternative Splicing, Western Blot, Flow Cytometry, Quantitative Proteomics, Activation Assay, Expressing

Journal: Cell reports

Article Title: Quantitative trait loci mapping provides insights into the genetic regulation of dendritic cell numbers in mouse tissues

doi: 10.1016/j.celrep.2024.114296

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After being blocked, the western blot membrane was subsequently incubated with anti-Gramd4 antibody (Clone C-8, Santa-Cruz).

Techniques: Marker, Staining, Recombinant, Red Blood Cell Lysis, Software

Journal: Cell Death Discovery

Article Title: Knockdown of SUCLG2 inhibits glioblastoma proliferation and promotes apoptosis through LMNA acetylation and the mediation of H4K16la lactylation

doi: 10.1038/s41420-025-02856-4

Figure Lengend Snippet: A H4K16la and L-Lac expression patterns in shNC and shSUCLG2 cells of U251 and LN229 were determined using western blotting. B Cell precipitates were collected for cleavage under targets and tagmentation (CUT&Tag) assay to identify H4K16la binding peaks, and the heatmap shows the distribution of H4K16la peaks near the translation start site (TSS). C Comparison of shNC and shSUCLG2 peaks in LN229 cells. D Genomic distribution of H4K16la, which was annotated for its localisation (promoter, exon, intron, or intergenic) and quantified in relative and absolute amounts. E Volcano plots of upregulated versus downregulated genes after CUT&Tag assay for H4K16la binding. F The heat map shows genes downregulated around TSS in LN229 cells containing shNC and shSUCLG2 as detected by the CUT&Tag assay. G KEGG analysis of CUT&Tag-detected downregulated genes at H4K16la. H GO analysis of CUT&Tag-detected downregulated genes. I Volcano plot of differential gene expression in RNA-seq. J KEGG analysis of downregulated genes in shSUCLG2 versus shNC using RNA-seq. K GO analysis of shSUCLG2 versus shNC downregulated genes using RNA-seq. L Combined CUT&Tag and RNA-seq analyses were performed to identify potential downstream target intersections of H4K16la. M Integrated genomics viewer trajectory track of CUT&Tag data showing enrichment of H4K16la at BEST1, GRAMD4H, and MBD6 promoter regions. N LN229 and U251 cells were treated with shNC or shRNA, and DNA fragments were immunoprecipitated with H4K16la antibody and analysed by qPCR. O Representative western blotting images show the quantification of BEST1, MBD6, and GRAMD4 protein levels. P Western blotting of IL-6 and IL-8 in shNC or shRNA lines of LN229 and U251. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: The following antibodies were used: SUCLG2 (A8976, 1:1 200, ABclonal, Wobum, MA), Bax (60267-1-Ig, 1:1000, Proteintech, Rosemont, IL), PCNA (HRP-60097, 1:1 000, Proteintech), Caspase-3 (68773-1-Ig, 1:1000, Proteintech), Bcl-2 (12789-1-AP, 1:1000, Proteintech), β-actin (11313-2-AP, 1:1 500, Proteintech), Cyclin D1 (60186-1-Ig, 1:1500, Proteintech), DLAT (ab172617, 1:1000, Abcam, Cambridge, UK), LMNA (ab172617, 1:1000, Abcam), L-Lac (PTM-1401RM, 1:1000, Jingjie Bio, Nanjing, China), D-Lac (PTM-1429RM, 1:1000, Jingjie Bio), HIF-1α (H1alpha67, 1:1000, Abcam), H4K16la (PTM-122, 1:1000, Jingjie Bio), H4 (PTM-1015RM, Jingjie Bio), Total oxidative phosphorylation Rodent Antibody Cocktail (ab110413, 1:1000, Abcam), BEST1 (1:1000, ab259836, Abcam); GRAMD4 (1:1000, 24299-1-AP9, Proteintech), MBD6 (1:1000, ab204403, Abcam); MFN1 (A21293, 1:1200, ABclonal); MFN2 (A19678, 1:1200, ABclonal); DR1 (A13298,1:1200, ABclonal); IL-6 (A26791, 1:1000, ABclonal); IL-8 (RP00052, 1:1000, ABclonal); anti-rabbit IgG (H + L) (ab205718, 1:5000, Abcam), and anti-mouse IgG (H + L) (ab205719, 1:5000, Proteintech).

Techniques: Expressing, Western Blot, Binding Assay, Comparison, Gene Expression, RNA Sequencing, shRNA, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 recruited the E3 ligase ITCH to target TAK1 for K48‐linked ubiquitination and degradation. (A) 293T cells were transfected with vector or GRAMD4‐Flag plasmid together with control‐siRNA or ITCH‐siRNAs and analysed for TAK1, ITCH and Flag‐tag expression by western blot. (B) HLF cells with vector and GRAMD4 overexpression were transiently transfected with control‐siRNA or ITCH‐siRNAs and analysed for TAK1, ITCH and GRAMD4 expression by western blot. (C) Co‐IP analysis of the binding between exogenous GRAMD4‐Flag and ITCH‐HA in co‐transfected HEK293T cells. (D) Immunoassay of lysates from HLF cells, followed by immunoprecipitation with an anti‐TAK1 antibody. Co‐IP assay of binding between TAK1 and ITCH was performed at exogenous (E) endogenous (F) levels when GRAMD4 was overexpressed or not. Immunoassay of 293T cells transfected with expression vectors for various combinations of AMD4‐Myc, TAK1‐Flag, HA‐Ub (WT)(G), Ub (K48)(H) and Ub (K63)(I) together with control‐siRNA or ITCH‐siRNA, followed by immunoprecipitation of lysates with an anti‐Flag antibody and immunoblot analysis with anti‐Myc, anti‐HA, anti‐Flag anti‐ITCH antibodies

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Transfection, Plasmid Preparation, FLAG-tag, Expressing, Western Blot, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: GRAMD4 inhibits tumour metastasis by recruiting the E3 ligase ITCH to target TAK1 for degradation in hepatocellular carcinoma

doi: 10.1002/ctm2.635

Figure Lengend Snippet: GRAMD4 exerted its tumour suppressive effects by modulating the protein levels of TAK1. (A) Western blot analysis of TAK1, p‐JNK, p‐p38, p‐ERK and p‐p65 protein levels in HLF cells stably transfected with GRAMD4 plasmid or vector control. (B) Western blot analysis of TAK1, p‐JNK, p‐p38, p‐ERK, and p‐65 protein levels in Hep3B cells stably transfected with lentivirus expressing Sh‐Ctrl, GRAMD4‐Sh1, or GRAMD4‐Sh2. (C) Western blot analysis of TAK1, p‐JNK, p‐p38, p‐ERK and p‐65 protein levels in GRAMD4‐depleted Hep3B cells transiently transfected with si‐NC, TAK1‐si. (D, E) The expression levels of MMP‐1, MMP‐3, MMP‐9, MMP‐10 and MMP‐13 in the indicated cells were analysed by qRT‐PCR. Experiments were performed in triplicate and data are shown as mean ± SD. (F) Transwell migration and invasion assays were performed with stably transfected HLF‐vector and HLF‐GRAMD4 cells. (G, H) Lung metastasis experiments were conducted in nude mice with the indicated stably transfected cells. Representative images of lung metastases (G) and H&E staining of lung tissues (H) are shown. Statistical analysis was performed using Student's unpaired t ‐test in (F), and the Mann–Whitney U test in (G). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies against GRAMD4 (24299‐1‐AP for WB, immunohistochemistry (IHC), immunofluorescence (IF) and (IP) and ITCH (20920‐1‐AP for WB, IP) were purchased from Proteintech (Wuhan, Hubei, China); primary antibodies against TAK1 (#5206 for WB, IHC, IF and IP), ERK (#4695 for WB), phospho‐ERK (#4370 for WB), p38 ((#8690 for WB), phospho‐p38 (#9215 for WB), JNK (#9252 for WB), phospho‐JNK(#9255 for WB) and Myc‐tag (#2276 for WB) were from CST (Danvers, MA, USA); Mouse serum IgG (I5381 for IP control), Rabbit serum IgG (I5006 for IP control), primary antibodies against Flag‐tag (F1804 for WB, IP and IF) and HA‐tag (H6908 for WB, IP and IF) were from Sigma (St. Louis, MO, USA); HRP‐conjugated anti‐Rabbit IgG (#111‐035‐003) and HRP‐conjugated anti‐Mouse IgG (#115‐035‐003) secondary antibodies were used for WB analysis.

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Migration, Staining, MANN-WHITNEY