alpha Tubulin Antibody - #AF4651

Figure 5 Characterization of the interaction between lncRNA-MEG3 and SUZ12. A) RNA-binding motif enrichment analysis showing the most frequently detected RNA-binding proteins associated with lncRNA-MEG3 by catRAPID (http://s.tartaglialab.com/page/catrapid_group). B) Fluorescent in situ hybridization (FISH) showing lncRNA-MEG3 localization (green) in primary mouse myoblasts and C2C12 myoblasts. Nuclei are stained with DAPI (blue) (n = 3). Scale bar = 20 µm. C–E) RNA pull-down assay of SUZ12, β-actin (negative control), and GAPDH (negative control) using antisense and sense probes for lncRNA-MEG3 in C2C12 myoblast lysates (n = 3). Input represents total lysates. F) Quantification of the lncRNA-MEG3 interaction with SUZ12 normalized to input level (n = 3). G) Predicted secondary structure of lncRNA-MEG3 highlighting structural domains (D1, D2, and D3). H) RNA pull-down assay was performed to analyze the binding of lncRNA-MEG3 WT, deletion expression vectors (D1, D2, and D3), and SUZ12 protein (n = 3). I,J) Domain mapping of SUZ12 using truncated fragments (B1, B2, and B3) in pull-down assays, identifying the lncRNA-MEG3 interaction domain in SUZ12 (n = 3). K) Schematic of the deletion constructs in the B3 domain, with the deleted regions located at the following amino acid positions: c1 (500-550 aa deletion), c2 (551–600 aa deletion), c3 (601–650 aa deletion), c4 (651–700 aa deletion), and c5 (701–739 aa deletion). L) RNA pull-down assay showing the interaction between biotinylated lncRNA-MEG3 and the B3 domain deletion variants (n = 3). M) Schematic representation of SUZ12 domains, indicating the lncRNA-MEG3 binding region and the EZH2/EED interaction region. N) Co-IP was performed to analyze the interaction between SUZ12 and EZH2/EED after C2C12 myoblasts were treated with RNase T1, DNase I, and lncRNA-MEG3 shRNA (n = 3). O) Co-IP was performed to analyze the interaction between SUZ12 and EZH2/EED after overexpression of lncRNA-MEG3 in C2C12 myoblasts (n = 3). P,Q) Co-IP was performed to analyze the interaction between SUZ12 and EZH2/EED in C2C12 myoblasts with knockdown of lncRNA-MEG3 (n = 3). R) IF staining of SUZ12 (red) in control and lncRNA-MEG3-overexpressing C2C12 cells showing enhanced nuclear localization. DAPI stains nuclei (blue) (n = 3). Scale bar = 20 µm. S) Western blot showing the expression of Suz12 in the nucleus, cytoplasm, and whole cell lysates of C2C12 myoblasts after lncRNA-MEG3 overexpression (n = 3). Lamin B1 was used as a marker for the nuclear protein, α-Tubulin as a marker for the cytoplasmic protein, and Gapdh as a marker for the total protein. Data are mean ± SD; p-values were calculated using Student's t-test. **p < 0.01.

Figure 2. C5a/C5aR pathway was related to breast cancer cell cuproptosis. (A) GO analysis of differently expressed genes in epithelial cells from the breast cancer group versus the normal group. (B) Violin plots show the cuproptosis score in 18 epithelial cell clusters. (C) Association between complement activation score (cited from BP entry in GO enrichment) and cuproptosis score analyzed by Spearman’s analysis. (D) ssGSEA analysis of cuproptosis in epithelial cells derived from tumor and normal tissue. (E) Cuproptosis score, complement activation score, and cells with both high scores visualized by UMAP plot of epithelial cells. (F) Potential trajectory of all epithelial cells identified two distinct cell fates colored by cluster. (G) Heatmap showing selected-related genes of cuproptosis and C5-C5aR pathway along the pseudotime, which was clustered into three profiles. Color keys differentially coding from blue to red indicated the relative expression levels from low to high. (H) Dot plots of dynamic expression of key genes in cuproptosis and C5-C5aR pathway along two cell fates. (I) PPI network shows potentially existing connections between complement-related genes, copper transporters genes and cuproptosis-related genes. (J) GSEA analysis of various forms of programmed cell death (PCD) in 4T1 cells treated with C5a. (K–L) Volcano plot and heatmaps of differentially expressed genes between control groups and C5a treated groups in 4T-1 cells. (M–N) Western blotting and RT-qPCR analysis of ATP7B expression in 4T1 cells between control and C5a treated groups. (O) GSEA analysis of various forms of PCD in MDA-MB-231 cells treated with C5a. (P–Q) Volcano plot and heatmaps of differentially expressed genes between control groups and C5a treated groups in MDA-MB-231 cells. (R–S) Western blotting and RT-qPCR analysis of ATP7B expression in MDA-MB-231 cells between control and C5a-treated groups. Statistical differences are assessed by unpaired t-test. Data were mean±SEM. *p

Fig. 3 Addition of P. pentosaceus YC inhibited Nlrc3 and promoted the gene expression of ISC markers. A KEGG enrichment analysis; B Heatmap of genes of NOD-like signaling pathway; C and D Volcano plot of DEGs in the compared groups (n = 3). E and F The relative gene expression of ISC markers (lgr5 and olfm4) in PI and DI; G The protein expression of Nlrc3 in PI and DI; H and I The quantification of the protein expression of Nlrc3 in PI and DI; J and K The relative gene expression of foxo3 and cyclinD1 in PI and DI (n = 6). Data are represented as mean ± SEM. Asterisk refers to the significant difference (ANOVA with Tukey’s test; *P 

FIGURE 4 UCP2 promoted NSCLC progression through the mTOR/HIF-1α pathway. (A) Western blotting assay underwent conduct after PC9, H1975, and A549 cell lines were transfected. (B) ImageJ software was used to quantify the protein expression. **p