human atat1 construct  (Addgene inc)

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