Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes a transcriptional downregulation of RAD51 and other proteins known to control DSB repair by HR. RNA-seq was performed on U2OS cells transfected with non-silencing (NS), NFKB2 or RELB siRNA. ( A ) Knockdown is efficient for both p100/52 and RelB, based on mRNA measurements by RNA-seq (left) and protein level by western blot (right). ( B ) KEGG pathway analysis of was performed to predict the 10 most upregulated and 10 most downregulated pathways in response to siNFKB2. Corresponding effects in response to siRELB are also displayed. ( C ) Expression levels are displayed for 39 genes with known relevance to HR, as defined by the KEGG database. Genes are arrayed from left to right based on degree of expression change after siNFKB2 (* denotes P < 0.01).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Control, RNA Sequencing, Transfection, Knockdown, Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 promotes downregulation of RAD51 protein. ( A ) Representative western blots demonstrate that transcriptional silencing of key NF-κB proteins leads to reduced levels of RAD51 protein. ( B ) Quantitation of western blots from three independent experiments demonstrate that RAD51 reductions after siNFKB2 are reproducible (error bars denote the SEM). ( C ) Comparable levels of RAD51 knockdown are observed using three different siRNAs (each at 25 nM) targeting NFKB2 in U2OS cells. ( D ) Forced overexpression of the p52 fragment of Nfkb2 in mouse lung tissue is associated with increased Rad51 expression (error bars denote standard error, * denotes P < 0.02, NS denotes non-silencing control).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Western Blot, Quantitation Assay, Knockdown, Over Expression, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Noncanonical NF-κB factor p100/p52 regulates homologous recombination and modulates sensitivity to DNA-damaging therapy

doi: 10.1093/nar/gkac491

Figure Lengend Snippet: Targeting of p100/p52 inhibits DNA damage-induced RAD51 focus formation in human cancer cells. Cells previously transfected with siRNA or non-silencing (NS) controls were pulse-labeled with EdU to mark S-phase cells and concomitantly treated with 30 nM CPT for 1 hour. Cells were harvested and immune-stained as indicated. Representative microscopic images of unselected nuclei ( A ) are displayed for cells collected 3-hours after CPT treatment, and ( B ) quantitation of RAD51 foci per nucleus is plotted for each of the indicated conditions. ( C ) Quantitation of EdU intensity in CPT-untreated cells is used to estimate cell cycle at the time of the EdU pulse. ( D ) A schematic representation of the treatment timeline is displayed. ( E ) The mean number of RAD51 foci per EdU-positive nucleus is plotted as a function of time following CPT treatment (error bars denote SEM for at least 100 nuclei within a single experimental replicate, * denotes P < 0.05, *** denotes P < 1e−13, and ns denotes P > 0.05).

Article Snippet: The following primary antibodies were used: mouse anti-human NF-κB1 p105/p50 (Santa Cruz 8414), mouse anti-human NF-κB2 p100/p52 (Millipore 05–361), rabbit anti-human RelB (Cell Signaling Technology 4922), rabbit anti-human RAD51 (Pacific Immunology, affinity purified from serum and used at 1:1000), mouse anti-human 53BP1 (Upstate 05-726 Clone BP13), rabbit anti-human H2A.X (Abcam ab229914), mouse anti-human γH2A.X (Millipore 05–636), rabbit anti-human BRCA2 (Bethyl A303-434A), rabbit anti-human GAPDH (Cell Signaling Technology 5174), and mouse anti-chicken α-tubulin (Fitzgerald 10R-T130A).

Techniques: Transfection, Labeling, Staining, Quantitation Assay

Journal: Oncology Letters

Article Title: Endocan silencing induces programmed cell death in hepatocarcinoma

doi: 10.3892/ol.2017.6857

Figure Lengend Snippet: The levels of LC3, ATG5, ATG7, DRAM and Beclin-1 protein expression were measured following transfection with endocan siRNA and normalized to β-actin. (A) Representative blot and (B) quantification of autophagy-associated protein expression. The expression levels of all the autophagy-associated proteins were significantly increased in endocan siRNA-treated cells compared with those in the NC and control groups. *P<0.05 in siRNA groups vs. NC groups. NC, negative control; siRNA, small interfering RNA; LC3, microtubule associated protein 1 light chain 3 α; ATG, autophagy related; DRAM, DNA damage regulated autophagy modulator 1.

Article Snippet: The primary antibodies were as follows: Endocan antibody (sc-515304; Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight; LC3 antibody (AL221; Beyotime Institute of Biotechnology; 1:500) at 4°C overnight; ATG5 antibody (PA2260; Boster Biological Technology, Pleasanton, CA, USA; 1:400,) at 4°C overnight; ATG7 antibody (PB9479; Boster Biological Technology; 1:400) at 4°C overnight; DRAM antibody (bs-4233R, BIOSS, Beijing, China; 1:500) at 4°C overnight; Beclin-1 antibody (AB123; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight and NF-κB p65 antibody (AF0246; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight.

Techniques: Expressing, Transfection, Control, Negative Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: Endocan silencing induces programmed cell death in hepatocarcinoma

doi: 10.3892/ol.2017.6857

Figure Lengend Snippet: Autophagy was detected using the mRFP-GFP-LC3 system. Red fluorescence, lysosomes; green fluorescence, autophagosome. mRFP-GFP-LC3 was transfected into cells of the (A) control, (B) endocan-targeting siRNA and (C) NC siRNA groups, and after 48 h expression was analyzed using fluorescence microscopy (magnification, ×100 or ×600). The mRFP-GFP-LC3 microtubule associated protein 1 light chain 3 α foci were monitored. Yellow fluorescence is the result of merging the red and green fluorescence images. Green fluorescent foci are observed to reduce alongside the formation of the autophagic lysosome. NC, negative control; siRNA, small interfering RNA. Scale bar=10 µm.

Article Snippet: The primary antibodies were as follows: Endocan antibody (sc-515304; Santa Cruz Biotechnology, Inc.; 1:200) at 4°C overnight; LC3 antibody (AL221; Beyotime Institute of Biotechnology; 1:500) at 4°C overnight; ATG5 antibody (PA2260; Boster Biological Technology, Pleasanton, CA, USA; 1:400,) at 4°C overnight; ATG7 antibody (PB9479; Boster Biological Technology; 1:400) at 4°C overnight; DRAM antibody (bs-4233R, BIOSS, Beijing, China; 1:500) at 4°C overnight; Beclin-1 antibody (AB123; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight and NF-κB p65 antibody (AF0246; Beyotime Institute of Biotechnology; 1:1,000) at 4°C overnight.

Techniques: Fluorescence, Transfection, Control, Expressing, Microscopy, Negative Control, Small Interfering RNA

Journal: PLoS ONE

Article Title: Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

doi: 10.1371/journal.pone.0080587

Figure Lengend Snippet: List of sense and anti-sense primers used for RT-PCR.

Article Snippet: For immunofluorescence microscopy, staining was performed using primary rabbit anti-human cytokeratin-14 (Covance Inc., Princeton, NJ); primary rabbit anti-human Stratifin antibody (Enzo Biomol, Framingdale, NY) and primary mouse anti-human involucrin (Abcam, Cambridge, MA) at 1∶1000 dilution and incubated overnight at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

doi: 10.1371/journal.pone.0080587

Figure Lengend Snippet: We evaluated ASC transdifferentiation into KLC by the expression of several keratinocyte markers such as, keratins, involucrin, stratifin and filaggrin. (a) mRNA expression of cytokeratin-5, cytokeratin-14, stratifin, involucrin and filaggrin in ASC, keratinocytes (K), fibroblasts (F), and KLC. Keratinocyte and fibroblasts were used as positive and negative control, respectively. The panel shows the absence with the exception of filaggrin of all other keratinocytes markers in ASC and fibroblasts, and the positive expression of these markers in KLC after ASC-transdifferentation, (Cytokeratin-5, 0.26±0.03 vs. 0.0008±0.0001; cytokeratin-14, 0.34±0.07 vs 0.001±0.000089; stratifin, 0.42±0.19 vs 0.000055±0.0000056 and involucrin, 1.3±0.9 vs 0.0008±0.0005; respectively). In the case of filaggrin, KLC showed almost a 3 fold up-regulation when compared to that of ASC (0.85±0.14 vs 0.29±0.01; respectively) **p<0.01; n = 3 independent experiments. Keratinocytes were normalized to 1 since these cells possess these markers. Statistical analysis was obtained comparing KLC to ASC. (b) Western blot analysis confirmed the mRNA findings. KLC showed the presence of cytokeratin-5, involucrin, filaggrin and stratifin proteins comparable to those from keratinocyte lysates, however no bands were detected in ASC or fibroblast lysates. (Arrows indicate Cytokeratin-5 band, according to its molecular weight. Upper bands may correspond to unspecific binding at a higher molecular weight not corresponding with cytokeratin-5 molecular weight) (Image representative n = 3 independent experiments).

Article Snippet: For immunofluorescence microscopy, staining was performed using primary rabbit anti-human cytokeratin-14 (Covance Inc., Princeton, NJ); primary rabbit anti-human Stratifin antibody (Enzo Biomol, Framingdale, NY) and primary mouse anti-human involucrin (Abcam, Cambridge, MA) at 1∶1000 dilution and incubated overnight at 4°C.

Techniques: Expressing, Negative Control, Western Blot, Molecular Weight, Binding Assay

Journal: PLoS ONE

Article Title: Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

doi: 10.1371/journal.pone.0080587

Figure Lengend Snippet: ASC were seeded in chamber slides and cultured in the presence of KCM. Cells were fixed and stained at different time points (Days 0, 7, 14, 28 and 42) for evaluation. (a) ASC were immunostained with anti-human cytokeratin-14. DAPI was used as counterstaining. Images demonstrate detection of cytokeratin-14 as early as day 7 post-treatment. Keratinocytes and ASC were used as positive and negative controls, respectively. (Image representative n = 3 independent experiments) (b) ASC were immunostained with anti-human Involucrin and anti-human Stratifin. DAPI was used as counterstaining. Expression of stratifin was detected starting at day 14. Expression of involucrin was observed starting at day 28. Keratinocytes and ASC were used as positive and negative controls, respectively. (Image representative n = 3 independent experiments). Quantification of transdifferentiated ASC into KLC. ASC cells were seeded on chamber slides and cultured in the presence of KCM. At different time points (Days 0, 7, 14, 28, and 42) cells were fixed and stained using anti-human cytokeratin-14, anti-human Stratifin and anti-human Involucrin. Slides were scanned using TissueFAXS system and quantified using TissueQuest software. Average of positive stained cells were divided by the average of total number of cells (nuclei counts) and multiplied by 100, to obtain the percentage. (*n = 3 slides per time point).

Article Snippet: For immunofluorescence microscopy, staining was performed using primary rabbit anti-human cytokeratin-14 (Covance Inc., Princeton, NJ); primary rabbit anti-human Stratifin antibody (Enzo Biomol, Framingdale, NY) and primary mouse anti-human involucrin (Abcam, Cambridge, MA) at 1∶1000 dilution and incubated overnight at 4°C.

Techniques: Cell Culture, Staining, Expressing, Software

Journal: Scientific Reports

Article Title: The effect of PDLIM7 on cell proliferation, migration, and drug sensitivity in clear cell renal cell carcinoma

doi: 10.1038/s41598-025-11495-9

Figure Lengend Snippet: PDLIM7 expression is increased in renal clear cell carcinoma tissue samples and cells and correlates with poor prognosis. ( A – C ) The public database shows elevated expression of PDLIM7 in ccRCC. ( D ) Immunohistochemical staining shows elevated expression of PDLIM7 in ccRCC. ( E ) PDLIM7 expression in ccRCC cells was significantly higher than that in normal cells HK2. Scale bar:10 µm and 5 µm ( F , G ) Effect of PDLIM7 on OS and PFS in ccRCC patients.

Article Snippet: PDLIM7 primary antibody(1:100,finetest,China), a DAB-enhanced chromogenic solution, and a rabbit two-step assay kit were obtained from Beijing Zhongsui Jinqiao Company.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: The effect of PDLIM7 on cell proliferation, migration, and drug sensitivity in clear cell renal cell carcinoma

doi: 10.1038/s41598-025-11495-9

Figure Lengend Snippet: PDLIM7 promotes cell proliferation, migration, and invasion in ccRCC. ( A ) Validation of western blot assay for transfection with sh-PDLIM7 in 769-p and Caki-1 cells. ( B ) Cloning assay for ccRCC cell viability. ( C ) Detection of cellular wound healing capacity by scratch assay. Scale bar:500 µm ( D ) Transwell assay for cell migration and invasion. Scale bar:10 µm ( E ) Western blot for EMT-related protein expression.

Article Snippet: PDLIM7 primary antibody(1:100,finetest,China), a DAB-enhanced chromogenic solution, and a rabbit two-step assay kit were obtained from Beijing Zhongsui Jinqiao Company.

Techniques: Migration, Biomarker Discovery, Western Blot, Transfection, Cloning, Wound Healing Assay, Transwell Assay, Expressing

Journal: Scientific Reports

Article Title: The effect of PDLIM7 on cell proliferation, migration, and drug sensitivity in clear cell renal cell carcinoma

doi: 10.1038/s41598-025-11495-9

Figure Lengend Snippet: KEGG/GO functional enrichment and drug sensitivity. ( A ) Functional enrichment of PDLIM7 and related genes KEGG/GO. ( B ) PDLIM7 and drug correlation.

Article Snippet: PDLIM7 primary antibody(1:100,finetest,China), a DAB-enhanced chromogenic solution, and a rabbit two-step assay kit were obtained from Beijing Zhongsui Jinqiao Company.

Techniques: Functional Assay