pax5 sirna  (Addgene inc)

Journal: Molecular Medicine

Article Title: Reciprocal METTL3-PAX5 regulation in maintaining B-cell identity and promoting B-cell hyperreactivity in SLE

doi: 10.1186/s10020-025-01295-2

Figure Lengend Snippet: MeRIP-seq Profiling of m6A Modification Across Naïve B cells, Activated B cells, and ASCs. A Pie chart showing the distribution of m6A sites in seven regions of naïve B cells, activated B cells, and ASCs. B Top 1 motif region of naïve B cells, activated B cells, and ASCs. C Metagene profiles of m6A site distribution along a normalized transcript containing three rescaled non-overlapping segments: 5′ UTR, CDS, and 3′ UTR in activated B cell vs naïve B cell group, ASC vs activated B cell group, and ASC vs naïve B cell group. D Pie chart showing the distribution of m6A sites in seven regions of activated B cell vs naïve B cell group, ASC vs activated B cell group, and ASC vs naïve B cell group. E Top 1 motif region of activated B cell vs naïve B cell group, ASC vs activated B cell group, and ASC vs naïve B cell group. F Genes with changes in both RNA levels and m6A levels when compared across B cells (activated B cell vs naïve B cell group, ASC vs activated B cell group, and ASC vs naïve B cell group). G Plots of m6A peaks for Pax5 and Bach2 in naive B cells with m6A-IP and Input groups. H Plots of m6A peaks for Pax5 , Bach2 , and Xbp1 in naive B cells, activated B cell, and ASC. I - K Functional enrichment analysis of differentially expressed genes among different B cells (activated B cell vs naïve B cell group, ASC vs activated B cell group, and ASC vs naïve B cell group)

Article Snippet: Raji B cells in the exponential phase were grown for 24 h and then transfected with METTL3 -siRNA or PAX5 -siRNA (Ribo Bio) for 48 h according to the manufacturer’s protocols.

Techniques: Modification, Functional Assay

Journal: Molecular Medicine

Article Title: Reciprocal METTL3-PAX5 regulation in maintaining B-cell identity and promoting B-cell hyperreactivity in SLE

doi: 10.1186/s10020-025-01295-2

Figure Lengend Snippet: METTL3 and PAX5 interacted to maintain B-cell identity. A WB analysis of PAX5 protein level following METTL3 knockdown in Raji B cells. B Flow cytometry analysis of PAX5 MFI after METTL3 knockdown in Raji cells. C M6A-RIP-qPCR assay showing three m6A modification sites on PAX5 mRNA. D M6A-RIP-qPCR assay showing that decreased m6A modification at PAX5 mRNA upon STM2457 treatment. E METTL3 RIP assay showing direct binding of METTL3 to PAX5 mRNA. F Actinomycin D assay to examine the effect of METTL3 on PAX5 mRNA stability. RNA decay assay. The half-live of PAX5 mRNA was detected by quantitative RT-PCR. The remaining mRNAs were normalized to t = 0. G Actinomycin D assay to examine the effect of METTL3 on PAX5 mRNA stability. Quantitative RT-PCR analysis of PAX5 mRNA abundance, relative expression was normalized to t = 0 of Si-NC cells. H WB analysis of METTL3 protein level following PAX5 knockdown in Raji B cells. I Flow cytometry analysis of METTL3 MFI after PAX5 knockdown in Raji cells. J EMSA showing PAX5 binding to the METTL3 gene promoter (METTL3 probe: −347 to −365). K Dual luciferase reporter assay detecting the effect of PAX5 on METTL3 promoter transcriptional activity. L Flow cytometry analysis of PAX5 expression in CD19 + B cells from peripheral blood of SLE patients and healthy controls. HC, n = 18; SLE, n = 18. M Correlation of METTL3 expression with PAX5 expression in CD19 + B cells of SLE patients Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, ***, p < 0.001, ****, p < 0.0001, by two-tailed unpaired Student’s t tests

Article Snippet: Raji B cells in the exponential phase were grown for 24 h and then transfected with METTL3 -siRNA or PAX5 -siRNA (Ribo Bio) for 48 h according to the manufacturer’s protocols.

Techniques: Knockdown, Flow Cytometry, Modification, Binding Assay, Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay, Activity Assay, Two Tailed Test

Journal: Molecular Medicine

Article Title: Reciprocal METTL3-PAX5 regulation in maintaining B-cell identity and promoting B-cell hyperreactivity in SLE

doi: 10.1186/s10020-025-01295-2

Figure Lengend Snippet: Schematic diagram showing the role of METTL3 in B-cell activation and its involvement in SLE. Elevated METTL3 and m6A levels were observed in B cells from SLE patients, correlating with disease activity. METTL3 stabilizes PAX5 mRNA through m6A modification, promoting PAX5 expression. In turn, PAX5 upregulates METTL3 in a positive feedback loop, sustaining B-cell activation. MeRIP-seq analysis revealed distinct m6A methylation patterns in B-cell subsets, which are critical for B-cell activation, contributing to SLE pathogenesis

Article Snippet: Raji B cells in the exponential phase were grown for 24 h and then transfected with METTL3 -siRNA or PAX5 -siRNA (Ribo Bio) for 48 h according to the manufacturer’s protocols.

Techniques: Activation Assay, Activity Assay, Modification, Expressing, Methylation

Journal: Journal of Translational Medicine

Article Title: Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop

doi: 10.1186/s12967-024-05290-9

Figure Lengend Snippet: miR-532-3p plays an inhibitory role in ccRCC by targeting PAX5. A The expression of miR-532-3p in RCC cells (769P, 786-O, A498, ACHN, Caki-1) and human renal cortical proximal convoluted tubule epithelial cells (HK2). B CCK8 assays demonstrated that cell proliferation was inhibited after transfected with miR-532-3p mimics. C Transwell assays demonstrated that cell migratory and invasive capacity was inhibited after transfected with miR-532-3p mimics. D FACS assays demonstrated that cell apoptosis was enhanced after transfected with miR-532-3p mimics. E Luciferase reporter assay in HEK293T cells co-transfected mimics miR- miR-532-3p or mimics NC and candidate genes of Luc-wild-type or Luc-mutant. F Western blot analysis indicated that miR-532-3p could down-regulate PAX5 expression in RCC cells. G Binding site of miR-532-3p and PAX5. (NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Article Snippet: For transient transfection, RiboBio (Guangzhou, China) synthesized small interfering RNA (siRNA) targeting PAX5 along with a scrambled control.

Techniques: Expressing, Transfection, Luciferase, Reporter Assay, Mutagenesis, Western Blot, Binding Assay

Journal: Journal of Translational Medicine

Article Title: Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop

doi: 10.1186/s12967-024-05290-9

Figure Lengend Snippet: Down-regulation of PAX5 suppresses proliferation, migration and invasion of RCC cells in vitro. A CCK8 assays demonstrated that cell proliferation was inhibited after PAX5 knockdown. B Transwell assays demonstrated that cell migratory and invasive capacity was inhibited after PAX5 knockdown. C FACS assays demonstrated that cell apoptosis was enhanced after PAX5 knockdown. D GSEA analysis in TCGA patients revealed that PAX5 may be involved in PI3K signaling. E Western blot analysis indicated that circSOD2 promotes the ccRCC progression through PI3K-AKT-mTOR pathway. ( A–C si-NC, referred to a scramble control of siRNA; si-1 & si-2, referred to siRNA targeting to PAX5 with different sequences; E sh-NC, referred to a scramble control of shRNA; sh-1 & sh-2, referred to shRNA targeting to circSOD2 with different sequences). (NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.)

Article Snippet: For transient transfection, RiboBio (Guangzhou, China) synthesized small interfering RNA (siRNA) targeting PAX5 along with a scrambled control.

Techniques: Migration, In Vitro, Knockdown, Western Blot, Control, shRNA

Journal: Journal of Translational Medicine

Article Title: Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop

doi: 10.1186/s12967-024-05290-9

Figure Lengend Snippet: circSOD2 rescues the tumor suppressive effect of miR-532-3p. A Cell proliferation ability of 786-O transfected with control vector, miR-532-3p mimics alone or circSOD2 overexpressed plus miR-532-3p mimics. B Cell migration and invasion abilities of 786-O transfected with control vector, miR-532-3p mimics alone or circSOD2 overexpressed plus miR-532-3p mimics. C Cell proliferation ability of 769p transfected with control vector, miR-532-3p inhibitor alone or circSOD2 shRNA plus miR-532-3p inhibitor. D Cell migration and invasion abilities of 769p transfected with control vector, miR-532-3p inhibitor alone or circSOD2 shRNA plus miR-532-3p inhibitor. E Apoptosis of 786-O transfected with control vector, miR-532-3p mimics alone or circSOD2 overexpressed plus miR-532-3p mimics (above), and apoptosis of 769p transfected with control vector, miR-532-3p inhibitor alone or circSOD2 shRNA plus miR-532-3p inhibitor (below). F Western blot of PAX5 and p-PI3K levels after 769p transfected with control vector, miR-532-3p inhibitor alone or circSOD2 shRNA plus miR-532-3p inhibitor (left), and 786-O transfected with control vector, miR-532-3p mimics alone or circSOD2 overexpressed plus miR-532-3p mimics (right). G The diagram of the signal pathway. (NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Article Snippet: For transient transfection, RiboBio (Guangzhou, China) synthesized small interfering RNA (siRNA) targeting PAX5 along with a scrambled control.

Techniques: Transfection, Control, Plasmid Preparation, Migration, shRNA, Western Blot

Journal: Journal of Translational Medicine

Article Title: Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop

doi: 10.1186/s12967-024-05290-9

Figure Lengend Snippet: PAX5 enhances the biosynthesis of circSOD2 in ccRCC cells. A Venn diagram shows the intersection of transcription factors predicted by the three databases. B PAX5 has two possible transcription factor binding sites in the promoter of circSOD2. C qRT-PCR showed that the expression of circSOD2 decreased with the knockdown of the expression of PAX5 in 786-O cells. D Schematic diagram of luciferase reporter vectors containing mutations at each of these two sites (S1-MUT-Luc and S2-MUT-Luc). E Luciferase assays proved the the expression of PAX5 significantly activated the site 1, rather than the site 2. F ChIP assay showed that PAX5 was enriched at site 1. (NS, nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

Article Snippet: For transient transfection, RiboBio (Guangzhou, China) synthesized small interfering RNA (siRNA) targeting PAX5 along with a scrambled control.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Knockdown, Luciferase

Journal: The Journal of Experimental Medicine

Article Title: Upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes

doi: 10.1084/jem.20192373

Figure Lengend Snippet: NFATc3 binds to the LRRC55 promoter and induces LRRC55 overexpression. (A) Screening of TFs binding to the LRRC55 promoter using a promoter-binding TF profiling plate array ( n = 3). Podocytes were treated with Ang II for 24 h to activate TFs. Nuclear extracts were prepared and incubated with TF binding oligo probe mix with or without an LRRC55 promoter DNA fragment. After spin separation of complexes from unbound free biotin-labeled oligos, TF-bound probes were eluted from the column and used for hybridization and analysis. (B) The level of LRRC55 mRNA in podocytes treated with Ang II, C/EBPβ siRNA, IRF-2 siRNA, NFATc3 siRNA, STAT4 siRNA, PAX5 siRNA, and ERα siRNA ( n = 3). (C) Western blot analysis of nuclear NFATc3 in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (D) Immunofluorescence staining of nuclear NFATc3 in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (E) Schematic of the NFATc3 binding sites in the upstream sequence of the LRRC55 promoter. (F) ChIP analysis of the binding between NFATc3 and the LRRC55 promoter in podocytes treated with Ang II (1 µM) for 24 h ( n = 3). (G) Schematic of the WT and mutated (MUT) LRRC55 promoter-luciferase reporter plasmids. (H) Normalized luciferase activity of reporter constructs in podocytes cotransfected with NFATc3 plasmid for 24 h ( n = 5). (I) RT-PCR analysis of LRRC55 in podocytes transfected with NFATc3 plasmid for 24 h ( n = 5). (J) Western blot analysis of LRRC55 in podocytes transfected with NFATc3 plasmid ( n = 3). (K) RT-PCR analysis of LRRC55 in podocytes treated with Ang II (1 µM) and NFATc3 siRNA for 24 h ( n = 5). (L) Western blot analysis of LRRC55 in podocytes treated with Ang II (1 µM) and NFATc3 siRNA for 24 h ( n = 3). Data shown are representative of three experiments. For statistical analysis, one-way ANOVA with Tukey’s post hoc test was used for B and K, and a two-tailed Student’s t test was used for H and I. ***, P < 0.001. Scale bar = 20 µm. Scram., scrambled.

Article Snippet: The transfection of the pREP-NFATc3 plasmid (11790; Addgene), LRRC55 siRNA (sc-96743, 15 nM), TRPC6 siRNA (sc-42672), C/EBPβ siRNA (sc-29229), IRF-2 siRNA (sc-35708), NFATc3 siRNA (sc-29413), STAT4 siRNA (sc-36568), PAX5 siRNA (sc-43996), and ERα siRNA (sc-29305) was conducted with Lipofectamine 2000 (11668–019; Invitrogen).

Techniques: Over Expression, Binding Assay, Incubation, Labeling, Hybridization, Western Blot, Immunofluorescence, Staining, Sequencing, Luciferase, Activity Assay, Construct, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Transfection, Two Tailed Test

Journal: Epigenetics

Article Title: Key tumor suppressor genes inactivated by “greater promoter” methylation and somatic mutations in head and neck cancer

doi: 10.4161/epi.29025

Figure Lengend Snippet: Figure 4. qMSP results for PAX1, PAX5, ZIC4 and PLCB1 . ( A ) Graphical expression of the logistic regression, Pr ( HNSCC = 1 ) = logit −1 (b 0 + b 1 x methylation ) in tissue from 76 participants with data overlain. The predictor methylation is the qMSP value for each case (1) and each control (0). Cutoff methylation values for PAX1, PAX5 ZIC4 and PLCB1 are shown by the vertical dotted line. Probability of HNSCC is shown in red; ( B ) Scatterplots of quantitative MSP analysis of candidate genes promoters in the Validation screen cohort, which consisted of 76 HNSCC tumor tissue samples and 19 normal tissue samples obtained from uvulopharyngopalatoplasty (UPPP) procedures performed in non-cancer patients. The relative level of methylated DNA for each gene in each sample was determined as a ratio of MSP for the amplified gene to ACTB and then multiplied by 1000 [(average value of duplicates of gene of interest / average value of duplicates of ACTB) x 1000] for, PAX1, PAX5, ZIC4, and PLCB1 . Red line denotes cutoff value; ( C ) Sensitivity, Specificity and AUC results for qMSP analysis; ( D ) Receiver Operator Characteristics (ROC) curve for promoter methylation of PAX1, PAX5, ZIC4 and PLCB1 genes in the validation cohort. The figure shows that for this four gene panel the qMSP results have 96% sensitivity, 94% specificity, a 0.97 AUC and a Positive Predictive Value of 98.5%.

Article Snippet: We knocked down PAX5 in the 22B cell line using the ON-TARGETplus Pool of siRNAs against PAX5 and a non-targeting Pool of siRNAs (Thermo Scientific) as control.

Techniques: Expressing, Methylation, Control, Biomarker Discovery, Amplification

Journal: Epigenetics

Article Title: Key tumor suppressor genes inactivated by “greater promoter” methylation and somatic mutations in head and neck cancer

doi: 10.4161/epi.29025

Figure Lengend Snippet: Figure 5. Proposed genomic and epigenomic interactions in HNSCC: ( A ) Proposed partial pathway interplay of p53 and PAX5 in HNSCC. Downregulation of PAX5 leads to differentiation. When methylated, PAX5 an upstream target of p53 , fails to activate the later which is also silenced due to mutations, and thus DNA repair, Apoptosis, and Growth Arrest pathways are inactive; ( B ) PAX1 - NOTCH1 interplay through crosstalk of Hedgehog and Notch pathways in cell differentiation and proliferation signals. Notch1 induces p21 expression, either directly through the canonical pathway or indirectly through Hes1 and NFAT activation, leading in both cases to cell cycle arrest. Active Notch1 targets either the Hox family or Hes1 . Hes1 is active and will block differentiation. The HOX family of transcription factors, downstream targets of Notch signaling, is frequently silenced, thus blocking the activation of PAX1 which is also downregulated in HNSCC and will not promote differentiation. PAX1 expression can also be induced by Shh through Gli2 , which is active. Finally, proliferation is promoted through Gli2 interaction with CCND1 and CCND2 .

Article Snippet: We knocked down PAX5 in the 22B cell line using the ON-TARGETplus Pool of siRNAs against PAX5 and a non-targeting Pool of siRNAs (Thermo Scientific) as control.

Techniques: Methylation, Cell Differentiation, Expressing, Activation Assay, Blocking Assay