Journal: BMC Bioinformatics

Article Title: CNstream: A method for the identification and genotyping of copy number polymorphisms using Illumina microarrays

doi: 10.1186/1471-2105-11-264

Figure Lengend Snippet: Quantitative PCR results . (a) ΔΔ C T values for 20 individuals for the TUSC3 locus, which clearly validate the deletions assigned by CNstream (red bars) versus the 2-copy samples (blue bars). (b) ΔΔCt values for the 20 individuals analyzed for the PLEC1 locus demonstrate no differences between those that were called with a deletion (in blue) and those that were called as 2-copy samples (in red) by PennCNV.

Article Snippet: For PennCNV validation, the Taqman assay "Hs00837735_cn" (closest to rs11136336 SNP probe, bp 145,079,175) and within the CN region described in DGV (chr8:145,064,090-145,740,218) was used.

Techniques: Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: ΔNp63α promotes radioresistance in esophageal squamous cell carcinoma through the PLEC-KEAP1-NRF2 feedback loop

doi: 10.1038/s41419-024-07194-4

Figure Lengend Snippet: A KYES450 cells were subjected to anti-KEAP1 (or anti-PLEC) immunoprecipitation (IP); the coprecipitating endogenous PLEC (or KEAP1) were examined by immunoblot analyses (IB). B KYSE450 cells were subjected to immunofluorescence (IF). C HEK-293T cells, expressing NRF2, Flag-tagged KEAP1 and/or HA-tagged PLEC, were subjected to anti-Flag immunoprecipitation (IP); the co-precipitating NRF2, HA-PLEC, Flag-KEAP1 were examined by immunoblot analyses (IB). D the interaction of NRF2 with KEAP1 were quantified on the base of KEAP1 immunoprecipitation. E Sequence alignment highlighting the putative Keap1-binding motif in PLEC from different species and those previously reported in NRF2 and PGAM5. F KYSE450 cells expressing HA-tagged PLEC WT , PLEC ΔEQGE , or PLEC EQGA , were subjected to anti-HA immunoprecipitation (IP); the co-precipitating KEAP1 was examined by immunoblot analyses (IB). G KYSE450 cells expressing HA-tagged PLEC WT , PLEC ΔEQGE , or PLEC EQGA , were subjected to anti-NRF2 immunoprecipitation (IP); the co-precipitating ubiquitin was examined by immunoblot analyses (IB). KYSE450 cells stably expressing a control shRNA (shC) or shRNA specific for p63 (shp63 #1) were infected with lentivirus expressing PLECs (wild-type and two mutants) or empty vector (EV), treated with irradiation, then subjected to immunoblot analyses ( H ), FACS assays ( I , left panel), or cell viability testing ( I , right panel). Results are presented as means ± SD from three independent experiments in triplicates. **P < 0.01.

Article Snippet: For promoter assays, a fragment of human PLEC promoter containing ΔNp63α putative binding sites (P1 and P2) was inserted into the Gluc-On promoter reporter vector (pEZX-PG04, GeneCopoeia, Guangzhou, China) and designated as PLEC-Gluc-WT.

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Expressing, Sequencing, Binding Assay, Ubiquitin Proteomics, Stable Transfection, Control, shRNA, Infection, Plasmid Preparation, Irradiation

Journal: Cell Death & Disease

Article Title: ΔNp63α promotes radioresistance in esophageal squamous cell carcinoma through the PLEC-KEAP1-NRF2 feedback loop

doi: 10.1038/s41419-024-07194-4

Figure Lengend Snippet: A The Pearson correlation coefficient (R value) and a two-tail probability test (P value) between TP63 and PLEC were analyzed based on TCGA database. KYSE30, KYSE150 or NCI-H520 cells stably expressing ΔNp63α (WT or R304W) were subjected to immunoblot analyses ( B ) and QPCR assays ( C ). KYSE180, KYSE450 or HCC95 cells stably expressing a control shRNA (shC) or two different shRNAs specific for p63 were subjected to immunoblot analyses ( D ) and QPCR assays ( E ). F TP63 chromatin immunoprecipitation (ChIP) sequencing data from human keratinocytes (GEO accession number GSE32061) identifies the putative TP63-binding sites within PLEC enhancer. G HEK-293T cells were co-transfected with PLEC-Gluc-SEAP reporter (WT, P1 Mut, P2 Mut or P1&P2 Mut) and ΔNp63α (WT or R304W) expression plasmid. PLEC-Gluc and SEAP activities in media were measured at 36 h post-transfection. H ChIP assays using indicated antibodies or a normal rabbit IgG were performed in KYSE450 cells. Primers specific for P1, P2, or NC (negative control) were used. The K14 was used as a positive control. Results are presented as means ± SD from three independent experiments in triplicates. **P < 0.01.

Article Snippet: For promoter assays, a fragment of human PLEC promoter containing ΔNp63α putative binding sites (P1 and P2) was inserted into the Gluc-On promoter reporter vector (pEZX-PG04, GeneCopoeia, Guangzhou, China) and designated as PLEC-Gluc-WT.

Techniques: Stable Transfection, Expressing, Western Blot, Control, shRNA, Chromatin Immunoprecipitation, ChIP-sequencing, Binding Assay, Transfection, Plasmid Preparation, Negative Control, Positive Control

Journal: Cell Death & Disease

Article Title: ΔNp63α promotes radioresistance in esophageal squamous cell carcinoma through the PLEC-KEAP1-NRF2 feedback loop

doi: 10.1038/s41419-024-07194-4

Figure Lengend Snippet: Our study demonstrates that ΔNp63α, which is overexpressed in ESCC, drives the transcriptional activation of PLEC. PLEC, in turn, binds competitively to KEAP1, preventing NRF2 from undergoing ubiquitin–proteasome degradation. This stabilization facilitates the nuclear translocation of NRF2, which enhances antioxidant capacity and promotes radioresistance. Additionally, low-dose irradiation activates NRF2, which subsequently upregulates ΔNp63α expression. Collectively, these findings suggest that ΔNp63α and NRF2 participate in a positive feedback loop, amplifying antioxidant responses and helping maintain cellular redox homeostasis, which ultimately contributes to enhanced radioresistance in ESCC.

Article Snippet: For promoter assays, a fragment of human PLEC promoter containing ΔNp63α putative binding sites (P1 and P2) was inserted into the Gluc-On promoter reporter vector (pEZX-PG04, GeneCopoeia, Guangzhou, China) and designated as PLEC-Gluc-WT.

Techniques: Activation Assay, Ubiquitin Proteomics, Translocation Assay, Irradiation, Expressing