control small interfering rna sirna  (Bioneer Corporation)

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: TME profile of tumor tissues from 30 CRC patients using scRNA-seq. (A) A total of 99,268 cells derived from scRNA-seq of tumor tissues from 30 CRC patients were clustered and annotated to identify main cell types, as displayed in UMAP. (B) Subclustering and annotation of T/NK cell subtypes. (C) Heatmap showing proportions of each cell type per patient, with MSI status, and APC , TP53 , and RAS mutation status, and bar graph displaying the total cell counts of the main cell types. (D) Violin plots showing proportions of CD4 + and CD8 + T cells according to TP53 and RAS mutation status (left), and proportions of CD4 + T cell subtypes according to TP53 mutation and CD8 + T cell subtypes according to RAS mutation (right). P values were obtained using the Wilcoxon rank-sum test. Red dots indicate the median. (E) Heatmap showing representative examples from the pathway enrichment analysis of DEGs from T cells according to MSI and mutation statuses . Abbreviations: Adj. P , adjusted P value; APC , adenomatosis polyposis coli; CRC, colorectal cancer; DEG, differentially expressed gene; MAIT, mucosal-associated invariant T cell; MSI, microsatellite instability; MSI-H, microsatellite instability-high; MSS, microsatellite stable; MUT, mutation; NF-κB, nuclear factor kappa B; NK, natural killer cell; RAS, rat sarcoma virus; scRNA-seq, single-cell RNA sequencing; T/NK, T cell/natural killer cell; Tcm, central memory T cell; Tem, effector memory T cell; Temra, terminally differentiated effector memory T cell; Tex, exhausted T cell; Tfh, T follicular helper cell; Th1, T helper 1; Th2, T helper 2; Th17, T helper 17 cell; TME, tumor microenvironment; Tnaive, naive T cell; TP53 , tumor protein 53; Treg, regulatory T cell; Trm, tissue-resident memory T cell; TNF-alpha, tumor necrosis factor-alpha; UMAP, uniform manifold approximation and projection; WT, wild type.

Article Snippet: To silence TP53 , gene-specific and control small interfering RNA were purchased from Bioneer and transfected into CRC cancer cell lines using Lipofectamine RNAiMax (13778075, Invitrogen) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Mutagenesis, Virus, Single Cell, RNA Sequencing

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: WT p53 suppresses mRNA and protein levels of C/EBPβ in CRC cell lines. (A) Protein and mRNA expression of C/EBPβ in CT26 cells stably expressing shCtrl or sh Trp53 were analyzed by Western blotting and real-time PCR, respectively. (B) C/EBPβ protein expression was determined in CRC cell lines harboring TP53 WT, nonsense mutation, and missense mutation treated with nutlin-3a (10 μmol/l) for WT p53 induction or vehicle for 24 h (left), as well as CEBPB mRNA levels in TP53 WT cell lines (right). (C) C/EBPβ protein expression was compared in TP53 WT HCT116 and SNU1544 cells after transfection with siCtrl or si TP53 , followed by nutlin-3a (10 μmol/l) or vehicle treatment for 24 h. (D) C/EBPβ protein expression was determined in TP53 nonsense SNUC1 and SNU1411 cells after transient transfection with EV or TP53 WT, followed by 24 h of nutlin-3a (10 μmol/l) treatment. (E) C/EBPβ protein levels were determined in SNU1544 cells pretreated with nutlin-3a (10 μmol/l) for 16 h and stimulated with 8-Br-cAMP (100 μmol/l), an inducer of CEBPB transcription, for 8 h. (F) C/EBPβ protein levels were determined in SNU1411 cells transiently transfected with EV, TP53 WT, or TP53 R175H for 24 h. (G) C/EBPβ protein (left) and mRNA (right) levels were assessed in p53-null Caco-2 cells stably transduced with doxycycline-inducible TP53 WT or TP53 R175H following treatment with 1 μg/ml doxycycline for 48 h. (H) C/EBPβ protein stability was compared in shCtrl and sh Trp53 CT26 cells after CHX treatment (5 μg/ml) for the indicated times. (I) SNU1544 were treated with nutlin-3a (10 μmol/l) for 21 h, followed by MG132 (2 μmol/l) for 3 h to block proteasomal degradation. Protein expression was analyzed by Western blotting, representative of 3 independent experiments, and the values presented underneath or in the graph (H) were generated by densitometric analyses using ImageJ. mRNA levels were determined using real-time PCR. The bar graphs represent the mean ± SD; each dot represents an independent biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: 8-Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; CEBPB , CCAAT enhancer binding protein beta; CHX, cycloheximide; CRC, colorectal cancer; doxy, doxycycline; EV, empty vector; SD, standard deviation; shCtrl, control short hairpin RNA; sh Trp53 , short hairpin RNA for Trp53 ; si TP53 , small interfering RNA for TP53 ; WT, wild type.

Article Snippet: To silence TP53 , gene-specific and control small interfering RNA were purchased from Bioneer and transfected into CRC cancer cell lines using Lipofectamine RNAiMax (13778075, Invitrogen) according to the manufacturer’s instructions.

Techniques: Expressing, Stable Transfection, Western Blot, Real-time Polymerase Chain Reaction, Mutagenesis, Transfection, Transduction, Blocking Assay, Generated, Binding Assay, Plasmid Preparation, Standard Deviation, Control, shRNA, Small Interfering RNA

Journal: The FASEB Journal

Article Title: Dissecting Shared Genetic Architecture of Thoracic Aortic Aneurysm and Aortic Related Traits and Identifying SplA /Ryanodine Receptor Domain and SOCS Box Containing 1 Involved in Smooth Muscle Phenotype Switching and Cell Senescence Through Alternative Splicing

doi: 10.1096/fj.202502457R

Figure Lengend Snippet: SplA/Ryanodine Receptor Domain and SOCS Box Containing 1 increases in thoracic aortic aneurysm models and thoracic aortic aneurysm patients. (A) Immunostaining for SplA/Ryanodine Receptor Domain and SOCS Box Containing 1 (SPSB1) in thoracic aortic aneurysm (TAA) patients ( n = 7 in TAA group, n = in non‐TAA group). The bar plot show quantification of the percentage of SPSB1 positive areas; (B) Western blot analysis for SPSB1 in TAA patients, β‐tubulin was used as loading control ( n = 4/groups); (C) Ultrasonic plot of β‐aminopropionitrile (BAPN)‐induced thoracic aortic pathology models, transverse aortic constriction (TAC) models, and corresponding control groups in the phase of end diastole. The bar plot shows the mean lumen diameter of ascending aorta of each group ( n = 17 in BAPN group, n = 15 in BAPN‐control group, n = 16 in TAC group, n = 15 in TAC sham‐operated group). (D) Real‐time quantitative polymerase chain reaction (qPCR) was used to determine the expression of SPSB1 in BAPN‐induced thoracic aortic pathology models, TAC models, and corresponding control groups ( n = 6 in BAPN group, n = 5 in BAPN‐control group, n = 6 in TAC group, n = 6 in TAC sham‐operated group). (E) Hematoxylin and eosin staining (H&E), Sirius Red staining, and Immunostaining in the cross‐sections of thoracic aorta ( n = 5 in BAPN group, n = 3 in BAPN‐control group, n = 4 in TAC group, n = 4 in TAC sham‐operated group). Immunostaining for SPSB1 was conducted in BAPN‐induced thoracic aortic pathology models, TAC models, and corresponding control groups. The bar plot shows quantification of the percentage of SPSB1 positive areas. Statistical analysis was performed using two‐tailed Student's t ‐test. * p < 0.05; ** p < 0.01; *** p < 0.001; (F) Western blot analysis for SPSB1 in BAPN models and control group, β‐tubulin was used as loading control ( n = 8 in BAPN group, n = 7 in control group); (G) Western blot analysis for SPSB1 in TAC models and control group, β‐tubulin was used as loading control ( n = 6 in TAC group, n = 6 in sham‐operated group. In the same group of mice, tissue were randomly pooled in pairs for protein extraction). SiSPSB1, SPSB1 knockdown with small interfering RNAs; ctrl, control group; sham, sham‐operated group.

Article Snippet: Small interfering RNAs (siRNAs) targeting SPSB1 were synthesized by MCE Corporation (Guangzhou, China).

Techniques: Immunostaining, Western Blot, Control, Real-time Polymerase Chain Reaction, Expressing, Staining, Two Tailed Test, Protein Extraction, Knockdown

Journal: The FASEB Journal

Article Title: Dissecting Shared Genetic Architecture of Thoracic Aortic Aneurysm and Aortic Related Traits and Identifying SplA /Ryanodine Receptor Domain and SOCS Box Containing 1 Involved in Smooth Muscle Phenotype Switching and Cell Senescence Through Alternative Splicing

doi: 10.1096/fj.202502457R

Figure Lengend Snippet: SplA/Ryanodine Receptor Domain and SOCS Box Containing 1 regulates smooth muscle cell phenotype switching and cell senescence. (A) SplA/Ryanodine Receptor Domain and SOCS Box Containing 1 (SPSB1) expression among distinct cellular populations; (B) SPSB1 was knockdown in aortic smooth muscle cell (SMC). The mRNA levels of α‐SMA, SM22, and KLF4 were detected by Real‐time quantitative polymerase chain reaction (qPCR) ( n = 6/experiments); (C) Immunofluorescent staining for α‐SMA (green), SM22 (green), KLF4 (green), and staining with DAPI (blue) in the aortic SMC after SPSB1 silenced; (D) Western blot analysis of the indicated proteins in SPSB1 knockdown aortic SMCs. β‐tubulin was used as a loading control for Western blotting ( n = 4/experiments). (E) Representative images of SA‐β‐gal (senescence‐associated β‐galactosidase)–stained SPSB1 knockdown aortic SMC and statistical analysis (right) ( n = 7 in control group, n = 5 in siRNA group). The blue regions are positively stained. Scale bar = 100 μm.; (F) Western blot analysis of senescence markers in SPSB1 knockdown aortic SMCs. β‐tubulin was used as a loading control for Western blotting ( n = 4/experiments); (G) Quantification of mRNA levels of senescence‐associated secretory phenotype (SASP) components in SPSB1 knockdown aortic SMCs ( n = 6/experiments). Statistical analysis was performed using two‐tailed Student's t ‐test; (H) Interleukin 6 (IL‐6) concentration in the culture supernatant of SPSB1 knockdown aortic SMCs. Statistical analysis was performed using two‐tailed Student's t ‐test ( n = 4/experiments); (I) The activity of MMP2 and MMP9 in culture medium was measured by gel zymography in SPSB1 knockdown aortic SMCs. SiSPSB1, SPSB1 knockdown with small interfering RNAs; ctrl, control group.

Article Snippet: Small interfering RNAs (siRNAs) targeting SPSB1 were synthesized by MCE Corporation (Guangzhou, China).

Techniques: Expressing, Knockdown, Real-time Polymerase Chain Reaction, Staining, Western Blot, Control, Two Tailed Test, Concentration Assay, Activity Assay, Zymography

Journal: The FASEB Journal

Article Title: Dissecting Shared Genetic Architecture of Thoracic Aortic Aneurysm and Aortic Related Traits and Identifying SplA /Ryanodine Receptor Domain and SOCS Box Containing 1 Involved in Smooth Muscle Phenotype Switching and Cell Senescence Through Alternative Splicing

doi: 10.1096/fj.202502457R

Figure Lengend Snippet: SplA/Ryanodine Receptor Domain and SOCS Box containing 1 regulates global alternative pre‐mRNA splicing profiles in aortic smooth muscle cells. (A) Gene Ontology (GO) enrichment analysis of SplA/Ryanodine Receptor Domain and SOCS Box Containing 1 (SPSB1) co‐expressed genes in contractile smooth muscle cells (SMC) in single‐cell RNA‐sequencing; (B) GO enrichment analysis of quantitative proteomics in SPSB1 knockdown aortic SMCs; (C) Heatmap showing percent spliced‐in (PSI) values for differentially spliced alternative splicing events (DAS) between SPSB1 knockdown aortic SMCs and control group ( n = 3/group); (D) Dot plot showing the distribution of PSI values for five AS types upon SPSB1 knockdown in aortic SMCs; (E) Bar plot of the DAS observed in RNA‐seq analysis of SPSB1 knockdown aortic SMCs and control group; (F) Schematic (right) and distribution (left) of five alternative splicing event types that differentially change upon SPSB1 knockdown aortic SMCs; (G) Representative Sashimi plot showing the decreased SEs of the RPL17, ATM, FOXM1, and MRPL43 genes in SPSB1 knockdown aortic SMCs and control group. SiSPSB1, SPSB1 knockdown with small interfering RNAs; ctrl, control group; SE, skipped exon; RI, retained intron; A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; MXE, mutually exclusive exon.

Article Snippet: Small interfering RNAs (siRNAs) targeting SPSB1 were synthesized by MCE Corporation (Guangzhou, China).

Techniques: RNA Sequencing, Quantitative Proteomics, Knockdown, Alternative Splicing, Control