Journal: Veterinary Sciences

Article Title: Roles of WNT6 in Sheep Endometrial Epithelial Cell Cycle Progression and Uterine Glands Organogenesis

doi: 10.3390/vetsci8120316

Figure Lengend Snippet: The effects of WNT6 transfection on β-catenin expression in EECs. ( A , B ) Relative expression of WNT6 and β-catenin protein to β-actin by WNT6 knockdown. ( C , D ) Relative expression of WNT6 and β-catenin protein to β-actin by WNT6 overexpression. si-WNT6, the small interfering RNA for WNT6 . pEX-4-WNT6, pEX-4 vector with WNT6 -CDs inserted. siRNA with scrambled sequence absent in the sheep genome and pEX-4 was severed as negative control for knockdown and overexpression, respectively. The values are presented as means ± SDs. Means with different letters indicate significant differences ( p < 0.05).

Article Snippet: β-actin , bs-0061R , Bioss, Beijing, China , _ , 1:2000 , Actins are highly conserved globular proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells. β-actin is the protein for internal reference..

Techniques: Transfection, Expressing, Over Expression, Small Interfering RNA, Plasmid Preparation, Sequencing, Negative Control

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: Expression levels of DPEP2 in generalized carcinoma and LUAD. (A) The heatmap of the top ten upregulated and downregulated genes with LUAD progression based on RNA sequencing data in Gene Expression Omnibus (GSE31210). (B) The expression level of DPEP2 in different tumors in TCGA was determined using TIMER2.0. (C) The cBioPortal OncoPrint map shows the distribution of DPEP2 genome changes in patients with LUAD. (D) Expression level of DPEP2 in normal tissues and tumor tissues. (E) Expression level of DPEP2 in normal tissues and the paired adjacent tumor tissues. (F) Transcriptional level of DPEP2 in the human lung bronchial epithelial cell line BEAS-2B and various LUAD cell lines (A549, H1650, and H1299). (G) Validation of the expression level of DPEP2 in LUAD using the Human Protein Atlas database (immunofluorescence). (H) The protein levels of DPEP2 were determined using western blotting in different LUAD cell lines. *P<0.05, **P<0.01 and ***P<0.001; two-tailed Student's t-test. DPEP2, dipeptidase-2; LUAD, lung adenocarcinoma.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: Expressing, RNA Sequencing, Gene Expression, Biomarker Discovery, Immunofluorescence, Western Blot, Two Tailed Test

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: DPEP2 affects migration, invasion, and EMT of lung adenocarcinoma cells. (A) Expression of DPEP2 in empty vector control (Vector) and DPEP2-overexpressing cells (DPEP2) was detected using western blotting and reverse transcription-quantitative PCR assays. GAPDH served as a loading control. (B) Representative images and quantitative analysis of wound-healing assay of A549 and H1650 cells (scale bar, 200 µm). (C and D) Transwell representative images and analysis of (C) cell migration and (D) invasion (scale bar, 100 µm). (E and F) The EMT markers (E-cadherin, N-cadherin, vimentin, and α-SMA) were analyzed using western blot analysis and IF staining (scale bar, 50 µm). Histograms represent the mean ± standard deviation based on three independent experiments. *P<0.05, **P<0.01 and ***P<0.001, two-tailed Student's t-test. DPEP2, dipeptidase-2; EMT, epithelial-mesenchymal transition.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: Migration, Expressing, Plasmid Preparation, Control, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Wound Healing Assay, Staining, Standard Deviation, Two Tailed Test

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: DPEP2 enhances lung adenocarcinoma cell sensitivity to cisplatin by regulating cancer stem cell transformation. (A and B) Expression of CD44 and CD133 was determined by western blotting and immunofluorescence staining (scale bar, 50 µm). (C) Representative images and analysis of sphere formation in A549 and H1650 cells (scale bar, 100 µm). (D) Representative images of colony formation assays of A549 and H1650 cells treated with various concentrations of cisplatin at 0, 2, 4 or 8 µg/ml. (E and F) Cell Counting Kit-8 assay was used to analyze the sensitivity of A549 and H1650 cells to various concentrations of cisplatin at 0, 2, 4 or 8 µg/ml. **P<0.01, two-way ANOVA with post hoc Dunnett's t-test. (G) Representative flow cytometric images of A549 and H1650 cells treated with 0 or 2 µg/ml cisplatin. **P<0.01 and ***P< 0.001, two-tailed Student's t-test. DPEP2, dipeptidase-2.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: Transformation Assay, Expressing, Western Blot, Immunofluorescence, Staining, Cell Counting, Two Tailed Test

Journal: Oncology Reports

Article Title: Dipeptidase‑2 is a prognostic marker in lung adenocarcinoma that is correlated with its sensitivity to cisplatin

doi: 10.3892/or.2023.8598

Figure Lengend Snippet: DPEP2 enhances the sensitivity of lung adenocarcinoma cells to cisplatin in vivo . (A) Flow chart of the in vivo experiment with nude mice. (B and C) Nude mice were subcutaneously injected with a vector or DPEP2-overexpressing A549 stable strain. On days 7 and 14, mice were injected with PBS or cisplatin (20 mg/m 2 ). Every 7 days, the tumor volume was measured with calipers. Tumor volume on day 28 was assessed using a two-tailed Student's t-test (vector vs. DPEP2; and vector + cisplatin vs. DPEP2 + cisplatin). *P<0.05 and ***P<0.001. (D and E) On day 28, tumors were excised and weighed. Representative tumors isolated from nude mice and average tumor weights. **P<0.01 and ***P<0.001, one-way ANOVA with post hoc Dunnett's t-test. (F and G) Immunohistochemical staining of DPEP2, epithelial-mesenchymal transition-associated genes (E-cadherin, N-cadherin, vimentin, and α-SMA), stem cell biomarkers (CD44 and CD133), and the proliferation marker Ki67 in tumors of mice injected with vector or DPEP2-overexpressing cells (scale bar, 100 µm). DPEP2, dipeptidase-2.

Article Snippet: A549 (cat. no. CRL-7909; NSCLC cell line; was initiated by explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male; a hypotriploid human cell line with the modal chromosome number of 66, occurring in 24% of cells; mutations: KRAS, STK11, TP53) and H1650 (cat. no. CRL-5883; human NSCLC cell line; this was a cell line exhibiting epithelial morphology that was isolated in 1987 from the lung tissue of a 27-year-old, male smoker with stage 3B, bronchoalveolar carcinoma; mutations: EGFR, TP53) cell lines were obtained from the American Type Culture Collection.

Techniques: In Vivo, Injection, Plasmid Preparation, Two Tailed Test, Isolation, Immunohistochemical staining, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress

doi: 10.3390/ijms22116018

Figure Lengend Snippet: ECM stiffness-dependent microtubule acetylation regulates ER stress response signaling. ( A ) Western blot analysis of acetylated and detyrosinated α-tubulin levels in MDA-MB-231 cells cultured on 0.5 kPa PAGs or culture dishes for 48 h. ( B ) Western blot analysis of acetylated and detyrosinated α-tubulin levels in MDA-MB-231 cells cultured on non-adherent or adherent plates for 48 h. ( C ) Western blot analysis of ATAT1 KD efficiency in sh ATAT1 #1- and #2-treated MDA-MB-231 cells compared to shMock-treated cells. ( D ) Transmission electron microscopy of shMock- and sh ATAT1 #1-treated MDA-MB-231 cells in the presence or absence of 20 ng/mL tunicamycin (TM) for 24 h. The ER is indicated by arrowheads. Scale bar, 500 nm. ( E ) Morphometric analysis of ER width in shMock- and sh ATAT1 #1-treated MDA-MB-231 cells ( n = 10). Statistical analysis was performed using one-way ANOVA followed by Tukey multiple comparison tests. One-way ANOVA, F 3, 36 = 63.35. ** p < 0.01, *** p < 0.001. ( F ) Western blot analysis of phospho-IRE1α, IRE1α, ATF6, phospho-PERK, PERK, BiP, acetylated α-tubulin, and α-tubulin in shMock- and sh ATAT1 #1-treated MDA-MB-231 cells treated with 20 ng/mL TM for the indicated times. GAPDH was analyzed as a loading control in all Western blot assays. ( G ) Quantification of relative expression in UPR and ER stress marker proteins shown in ( F ). Band intensities of target proteins were quantified by densitometry using a Quantity One ® system. Relative expression of phospho-IRE1α, ATF6, and phospho-PERK were normalized by the band intensities of total IRE1a, full ATF6, and total PERK, respectively. The relative expression of Bip was normalized with GAPDH band intensity. The Western blot images are representative images of the results of at least three independent biological replicates.

Article Snippet: The coding sequence (CDS) of ATAT1 was amplified by PCR from a human ATAT1 construct (pEF5B-FRT-GFP-αTAT1; Addgene, Cambridge, MA, USA, #27099).

Techniques: Western Blot, Cell Culture, Transmission Assay, Electron Microscopy, Comparison, Control, Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress

doi: 10.3390/ijms22116018

Figure Lengend Snippet: Microtubule acetylation induces cancer-related gene expression through the alleviation of ER stress in breast cancer cells. ( A ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs upregulated in cells grown on a stiff matrix. The x -axis indicates the gene ratio, i.e., the ratio of DEGs in the given gene ontology (GO) term. The y -axis indicates KEGG pathways. Dot size represents the number of genes in each KEGG pathway. ( B ) Heatmap of 17 genes related to “Pathways in cancer” obtained through KEGG pathway analysis in mock and ATAT1 KO MDA-MB-231 cells. Genes with reduced expression in ATAT1 KO compared to mock-treated cells are indicated by blue letters. ( C ) mRNA levels of “Pathways in cancer” genes were decreased in ATAT1 KO MDA-MB-231 cells treated with 20 ng/mL tunicamycin for 24 h as indicated by RT-qPCR. Values represent the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001, N.S. not significant (Student’s t -test). ( D ) mRNA levels of seven genes that were downregulated upon tunicamycin treatment shown in ( C ) in ATAT1 KD compared to shMock-treated MDA-MB-231 cells as determined by RT-qPCR. Values represent the mean ± SD of three independent experiments. ** p < 0.01, *** p < 0.001, N.S. not significant (Student’s t -test). ( E ) mRNA levels of “Pathway in cancer” genes in control and ATAT1 overexpression lines after tunicamycin treatment as assessed by RT-qPCR. Values represent the mean ± SD of three independent experiments. Statistical significance of the differences in gene expression according to tunicamycin treatment in each of the cell lines transfected with empty vectors (EV) and ATAT1 overexpression (OE) vectors was analyzed by one-way ANOVA followed by Tukey multiple comparison tests ( # p < 0.05, ### p < 0.001). One-way ANOVA, F 2, 6 = 46.21 ( RASSF1 ), F 2, 6 = 12.70 ( MAPK8 ), F 2, 6 = 38.36 ( BCL2 ), F 2, 6 = 9.008 ( CXCL8 ), and F 2, 6 = 10.71 ( FGF1 ). Statistical significance of the differences between EV controls and ATAT1 OE cells was also analyzed using Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001). ( F ) Western blot analysis of calnexin (CANX), BiP, and acetylated α-tubulin in EV controls or ATAT1 OE cells after treatment with 20 ng/mL tunicamycin for 24 h. GAPDH was used as loading control. ( G ) Comparison of the number of invading cells via a Transwell invasion assay in cells cultured under the same conditions as in ( F ). Values represent the mean ± SD of three independent experiments. One-way ANOVA, F 2, 6 = 158.4. *** p < 0.001.

Article Snippet: The coding sequence (CDS) of ATAT1 was amplified by PCR from a human ATAT1 construct (pEF5B-FRT-GFP-αTAT1; Addgene, Cambridge, MA, USA, #27099).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Control, Over Expression, Transfection, Comparison, Western Blot, Transwell Invasion Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress

doi: 10.3390/ijms22116018

Figure Lengend Snippet: Focal adhesion assembly is regulated by microtubule acetylation and ER stress. ( A ) Functional annotation of 389 DEGs in ATAT1 KO MDA-MB-231 cells using PANTHER gene ontology (GO). ( B ) Heatmap showing genes related to focal adhesion assembly based on RNA-seq data from ATAT1 KO cells. ( C ) Validation of gene expression using Western blotting. shMock-treated cells treated or not with tunicamycin, and sh ATAT1 -treated cells were cultured for 24 h. Cell lysates were used for Western blot analysis of vinculin, FAK, talin, and acetylated α-tubulin, using GAPDH as a loading control. ( D ) Immunocytochemistry analysis of focal adhesions using antibodies against vinculin and F-actin in shMock-treated and ATAT1 KO cells cultured in the presence of 20 ng/mL tunicamycin for 24 h. Scale bar, 30 μm. ( E ) Paxillin-GFP-expressing shMock- and sh ATAT1 #1-treated MDA-MB-231 cells were starved for 16 h in serum-free RPMI1640 medium and then stimulated with 10% FBS with or without 20 ng/mL tunicamycin. Merged paxillin–GFP images in shMock- and sh ATAT1 #1-treated cells at 0 and 30 min. Red represents retracting focal adhesions and green represents newly forming focal adhesions. Scale bar, 10 μm. ( F ) Ratios of gain and loss of focal adhesions to total focal adhesion area. Values represent the mean ± SD of two independent experiments. Statistical significances were analyzed using one-way ANOVA followed by Tukey multiple comparison tests. One-way ANOVA (Loss; F 2, 3 = 15.98. * p < 0.05, Gain; F 2, 3 = 174.1, ** p < 0.01).

Article Snippet: The coding sequence (CDS) of ATAT1 was amplified by PCR from a human ATAT1 construct (pEF5B-FRT-GFP-αTAT1; Addgene, Cambridge, MA, USA, #27099).

Techniques: Functional Assay, RNA Sequencing, Biomarker Discovery, Gene Expression, Western Blot, Cell Culture, Control, Immunocytochemistry, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress

doi: 10.3390/ijms22116018

Figure Lengend Snippet: Expression levels of ATAT1 and ER stress marker genes are negatively correlated in breast cancer patients. ( A ) Representative images of immunohistochemistry of acetylated α-tubulin in normal, cancer-adjacent normal, and invasive carcinoma breast tissues. Scale bar, 200 μm. Lower panels, sample distribution by acetylated α-tubulin staining intensity. Staining intensity was marked (0 = absence, 1 = weak, 2 = moderate, 3 = strong). Values represent the mean ± SD of three independent experiments. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s multiple comparison tests (* p < 0.05, ** p < 0.01). One-way ANOVA (F 2, 47 = 8.552, p < 0.001). ( B ) Analysis of ATAT1 expression levels in normal breast, ductal breast carcinoma in situ, invasive ductal breast carcinoma, invasive lobular breast carcinoma, and invasive mixed breast carcinoma tissues using the Oncomine database. N, normal breast; DCIS, Ductal breast carcinoma in situ; IDBC, invasive ductal breast carcinoma; ILBC, invasive lobular breast carcinoma; IMBC, invasive mixed breast carcinoma. ( C ) Pearson’s correlations between mRNA levels of ATAT1 and ER stress marker genes in breast cancer patients based on the bc-GenExMiner RNA-seq dataset ( n = 4712). Number in the box indicates correlation coefficient value. ( D ) Expression levels of ATAT1 and ER stress marker genes in low-, medium-, and high-risk groups in 962 breast cancer patients from a TCGA dataset. ( E ) Kaplan–Meier plots of breast cancer patients based on the expression of ATAT1 and MAPK8, RASSF1, BCL2, CXCL8, and FGF1 in SurvExpress data ( n = 295). HR, hazard ratio.

Article Snippet: The coding sequence (CDS) of ATAT1 was amplified by PCR from a human ATAT1 construct (pEF5B-FRT-GFP-αTAT1; Addgene, Cambridge, MA, USA, #27099).

Techniques: Expressing, Marker, Immunohistochemistry, Staining, Comparison, In Situ, RNA Sequencing