Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Evaluation of pharmacodynamic responses to cancer therapeutic agents using DNA damage markers

doi: 10.1158/1078-0432.CCR-18-2523

Figure Lengend Snippet: (A) Baseline expression of selected DDR markers was demonstrated and quantified in a colorectal tissue array with 32 tumors (2 cores per case are included) and 15 normal colon tissues. At least 500 individual tumor nuclei were quantified in 95% of the tissue microarray cores. (B-C) Baseline marker quantitation in advanced stage colorectal cancers from patients enrolled in Phase 1 clinical trials at NCI. Median expression and inter-quantile range is indicated for each marker. A minimum of 4,000 individual tumor nuclei were quantitated across at least two nonadjacent slides per biopsy specimen. (D) Baseline expression in human colon adenocarcinoma patient-derived xenografts. At least 5,000 nuclei quantified per model. (E) Baseline expression of selected DDR markers was demonstrated and quantified in 8 colorectal cancer cell lines. Over 1000 individual nuclei were quantified for each cell line. (F) Inter-lesion baseline Rad51 quantitation from patients with advanced stage cancers with two biopsies each collected from the same lesion. *p< 0.05. The dashed line represents our empirically determined baseline value cutoff of 5% of cells ≥ 5 Rad51 foci per nucleus.

Article Snippet: Baseline biological variability was established for each biomarker across 64 individual cores from 32 colorectal (CRC) tumor resections contained in a paraffin-embedded colorectal tissue microarray (TMA; Indivumed, Hamburg, Germany).

Techniques: Expressing, Microarray, Marker, Quantitation Assay, Derivative Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Evaluation of pharmacodynamic responses to cancer therapeutic agents using DNA damage markers

doi: 10.1158/1078-0432.CCR-18-2523

Figure Lengend Snippet: Representative H&E and immunofluorescence (IFA) images from 3 patients with advanced colorectal cancer enrolled in NCI trial NCT01851369 before and 5 days after start of treatment with a DNA damaging therapeutic regimen consisting of TCR102 plus temozolomide administered orally once daily. Red scale bar represents 10 μm.

Article Snippet: Baseline biological variability was established for each biomarker across 64 individual cores from 32 colorectal (CRC) tumor resections contained in a paraffin-embedded colorectal tissue microarray (TMA; Indivumed, Hamburg, Germany).

Techniques: Immunofluorescence

Journal: Cell Death & Disease

Article Title: LncRNA CDKN2B-AS1 stabilized by IGF2BP3 drives the malignancy of renal clear cell carcinoma through epigenetically activating NUF2 transcription

doi: 10.1038/s41419-021-03489-y

Figure Lengend Snippet: a Pearson correlation analysis of the relationship between CDKN2B-AS1 and IGF2BP3 levels in TCGA-KIRC dataset. Normal tissues are shown as blue circles ( n = 72) and tumor tissues as red circles ( n = 533). b – e 769-P and ACHN cells were treated with si-IGF2BP3 for 48 h; qRT-PCR analysis of IGF2BP3, CDKN2B-AS1, and NUF2 expression with β-actin as the internal control ( b ); western blot analysis of IGF2BP3 and NUF2 protein levels with GAPDH as the internal control ( c ); qRT-PCR analysis of CDKN2B-AS1 ( d ) and NUF2 mRNA ( e ) stability, RNA levels were measured after treatment with actinomycin D (5 μg/mL) for 0, 2, 4, 8, 12, and 24 h, respectively, and compared to the level at 0 h. f RIP assay analysis of IGF2BP3 enrichment in CDKN2B-AS1, NUF2, and Linc01138 in 769-P and ACHN cells; RIP enrichment was measured using qRT-PCR normalized by the input RNA. g Colony formation assays were performed to assess cell proliferation in 769-P and ACHN cells treated with si-IGF2BP3. h Transwell assays were used to evaluate migration and invasion in 769-P and ACHN cells transfected with si-IGF2BP3. Scale bar: 50 μm. i , j Immunostaining score of IGF2BP3 in immunohistochemistry of patients with a different histologic grade ( i ) and TNM stage ( j ) from the paraffin-embedded KIRC tissue microarray (No. HkidE180Su02). k , l Kaplan–Meier analysis showed the overall survival and disease-free survival in KIRC patients based on the expression of IGF2BP3 in TCGA dataset; the high and low expression groups were divided by the median of IGF2BP3 levels. Error bars represent the SEM from at least three independent experiments, ** p < 0.01 vs. si-NC or anti-IgG, * p < 0.05 vs. si-NC or anti-IgG. ns. no significance ( p > 0.05). P value calculated by independent sample t test ( b – j ), or log-rank test ( k , l ).

Article Snippet: Paraffin-embedded KIRC tissue microarrays (No. HkidE180Su02) were purchased from Shanghai Biochip Company Ltd. (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Migration, Transfection, Immunostaining, Immunohistochemistry, Microarray

Journal: Cancer Medicine

Article Title: CUL4B promotes aggressive phenotypes of HNSCC via the activation of the Wnt/β‐catenin signaling pathway

doi: 10.1002/cam4.1960

Figure Lengend Snippet: Expression of CUL4B in human HNSCC. A, qRT‐PCR analysis of CUL4B mRNA in HNSCC tissues (tumor) and paired normal tissues (normal). B, qRT‐PCR analysis of CUL4B mRNA in male and female HNSCC tissues. C, CUL4B protein level in HNSCC tissues and paired normal tissues were assessed by Western blotting. D, Immunohistochemistry of CUL4B in nontumor and primary HNSCC tissue arrays. Scale bar: 50 μm. E, Kaplan‐Meier analysis of overall survival for patients with HNSCC. The analyses were conducted based on the immunohistochemistry of CUL4B and the survival information provided by the supplier. * P < 0.05

Article Snippet: The human HNSCC tissue microarrays (paraffin‐embedded), including follow‐up survival information, were provided by SuperBioChips.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cancer Medicine

Article Title: CUL4B promotes aggressive phenotypes of HNSCC via the activation of the Wnt/β‐catenin signaling pathway

doi: 10.1002/cam4.1960

Figure Lengend Snippet: Effects of CUL4B on malignant phenotypes in HNSCC cells. A, Cell viability of JHU‐012 cells transfected with vector or CUL4B was analyzed by MTS assays at different time points. B, Cell viability of sh con or sh CUL4B JHU‐012 cells was analyzed by MTS assays at different time points. C, Effects of CUL4B overexpression on anchorage‐dependent colony formation. Scale bar: 10 mm. D, Effects of CUL4B knockdown on anchorage‐dependent colony formation. Scale bar: 10 mm. E, CUL4B overexpression regulated transwell cell invasion and F, Matrigel invasion. Scale bar: 50 μm. G, CUL4B knockdown regulated transwell cell invasion and H, Matrigel invasion. Scale bar: 50 μm. I, Expression of epithelial markers, E‐cadherin and ZO‐1, and mesenchymal markers, vimentin, was analyzed by Western blotting in vector or CUL4B‐transfected JHU‐012 cells. J, Expression of epithelial markers, E‐cadherin and ZO‐1, and mesenchymal markers, vimentin, was analyzed by Western blotting in sh con or sh CUL4B JHU‐012 cells. * P < 0.05; ** P < 0.01

Article Snippet: The human HNSCC tissue microarrays (paraffin‐embedded), including follow‐up survival information, were provided by SuperBioChips.

Techniques: Transfection, Plasmid Preparation, Over Expression, Knockdown, Expressing, Western Blot

Journal: Cancer Medicine

Article Title: CUL4B promotes aggressive phenotypes of HNSCC via the activation of the Wnt/β‐catenin signaling pathway

doi: 10.1002/cam4.1960

Figure Lengend Snippet: CUL4B enhances Wnt/β‐catenin signaling in HNSCC cells. A, mRNA level of β‐catenin, cyclin D1, c‐myc, and MMP7 in vector or CUL4B overexpressed JHU‐012 cells was determined by qRT‐PCR. B, mRNA level of β‐catenin, cyclin D1, c‐myc, and MMP7 in sh con or sh CUL4B JHU‐012 cells was determined by qRT‐PCR. C, Protein level of β‐catenin, cyclin D1, c‐myc, and MMP7 in the indicated group was determined by qRT‐PCR and Western blotting. (D) Levels of the active form of β‐catenin in JHU‐012 cells with knockdown or overexpression of CUL4B. E and F, The activity of TCF/β‐catenin reporter (TOP/FOP Flash) in CUL4B‐knockdown (E) and CUL4B‐overexpressing cells (F). * P < 0.05; ** P < 0.01

Article Snippet: The human HNSCC tissue microarrays (paraffin‐embedded), including follow‐up survival information, were provided by SuperBioChips.

Techniques: Plasmid Preparation, Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Activity Assay

Journal: Cancer Medicine

Article Title: CUL4B promotes aggressive phenotypes of HNSCC via the activation of the Wnt/β‐catenin signaling pathway

doi: 10.1002/cam4.1960

Figure Lengend Snippet: Wnt/β‐catenin signaling regulates CUL4B functions in HNSCC cells Effects of XAV‐939, on CUL4B‐enhanced cell proliferation (A), migration (B), and invasion (C). Scale bar: 50 μm. Cells were treated with XAV‐939 or DMSO during the migration and invasion assays. ** P < 0.01

Article Snippet: The human HNSCC tissue microarrays (paraffin‐embedded), including follow‐up survival information, were provided by SuperBioChips.

Techniques: Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: Overexpression of the mitochondrial anti-viral signaling protein, MAVS, in cancers is associated with cell survival and inflammation

doi: 10.1016/j.omtn.2023.07.008

Figure Lengend Snippet: MAVS overexpression in diverse types of cancer (A–C) Formalin-fixed and paraffin-embedded US-Biomax tissue microarray slides containing the indicated tissues from cancer patients (n = 20) and normal tissue (n = 5) were immunohistochemistry stained using anti-MAVS antibodies and hematoxylin stained, as described in . Representative images from sections of the different indicated tumors and corresponding healthy tissues are shown. The slides were incubated overnight at 4°C with anti-MAVS antibodies in PBS containing 1% BSA and then with secondary antibodies in PBS containing 1% BSA. The slides were subsequently treated with 3′3-diaminobenzidine tetra-hydrochloride (DAB) and counterstained with hematoxylin. Negative controls without primary antibody incubation were also performed. Sections of tissue were observed under an Leica microscope, and images were taken at 200× magnification with the same light intensity and exposure time. The percentage of the tumor sections stained at the intensity indicated on the scale above are presented. (D) Peripheral blood mononuclear cells (PBMCs) were obtained from chronic lymphocytic leukemia (CLL) patients (n = 16) and healthy donors (n = 13) using Ficoll-Paque PLUS (GE Healthcare, Israel) density gradient centrifugation, as described previously. Representative immunoblots of cell lysates of PBMCs derived from CLL patients and healthy donors subjected to SDS-PAGE and immunoblotting using anti-MAVS and anti-actin antibodies are shown. (E) Representative immunoblots using anti-MAVS antibodies of tissue lysates of lung cancer samples from tumor tissue (T, n = 22) and healthy tissue (H, n = 22), each derived from the same lung of a lung cancer patient. (F) Quantification of MAVS levels in CLL and lung cancer patients relative to healthy donors presented as fold change. The results are the mean ± SD. (G) HEK-293, Hela, SHSY-5Y, PC3, A549, MDA-MB-231, and UMUC3 cell lines were grown in the appropriate medium, and cell lysates (10 μg of protein) were subjected to SDS-PAGE and immunoblotting with anti-MAVS and citrate synthase (CS) antibodies (n = 3). (H) Quantitative analysis of MAVS and CS expression levels in the different cell lines were presented relative to the level in SHSY-5Y cells for MAVS and to A549 cells for CS. (I) T-REx-293 cells were transiently transfected with pcDNA3 or MAVS-pcDNA3 using metafectene as in the section. MAVS expression was analyzed by immunoblotting 48 h post transfection. (J) Schematic presentation of the bicistronic nature of the MAVS transcript depicting met1 (methionine1) and met142 (methionine142) as the two translation initiation sites.

Article Snippet: MAVS overexpression in diverse types of cancer (A–C) Formalin-fixed and paraffin-embedded US-Biomax tissue microarray slides containing the indicated tissues from cancer patients (n = 20) and normal tissue (n = 5) were immunohistochemistry stained using anti-MAVS antibodies and hematoxylin stained, as described in .

Techniques: Over Expression, Microarray, Immunohistochemistry, Staining, Incubation, Microscopy, Gradient Centrifugation, Western Blot, Derivative Assay, SDS Page, Expressing, Transfection