human atat1 construct (Addgene inc)
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Structured Review
Addgene inc
human atat1 construct
Human Atat1 Construct, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+atat1+construct/pEF5B-FRT-GFP-%CE%B1TAT1+(Plasmid+%2327099)/pmc08199658-159-12-16
Average 93 stars, based on 23 article reviews
Human Atat1 Construct, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+atat1+construct/pEF5B-FRT-GFP-%CE%B1TAT1+(Plasmid+%2327099)/pmc08199658-159-12-16
Average 93 stars, based on 23 article reviews
human atat1 construct - by Bioz Stars,
2026-10
93/100 stars
Images
1) Product Images from "Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress"
Article Title: Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress
Journal: International Journal of Molecular Sciences
doi: 10.3390/ijms22116018
Figure Legend 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.
Techniques Used: Western Blot, Cell Culture, Transmission Assay, Electron Microscopy, Comparison, Control, Expressing, Marker
Figure Legend 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.
Techniques Used: Gene Expression, Expressing, Quantitative RT-PCR, Control, Over Expression, Transfection, Comparison, Western Blot, Transwell Invasion Assay, Cell Culture
Figure Legend 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).
Techniques Used: Functional Assay, RNA Sequencing, Biomarker Discovery, Gene Expression, Western Blot, Cell Culture, Control, Immunocytochemistry, Expressing, Comparison
Figure Legend 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.
Techniques Used: Expressing, Marker, Immunohistochemistry, Staining, Comparison, In Situ, RNA Sequencing
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Opto-αTAT1 (αTAT1-mCh-CIBN-NLS-LEXY) was constructed by using the HiFi DNA assembly method. αTAT1, mCherry, CIBN and LEXY (LOV2-NES) were amplified by standard PCR from Article Title: Microtubule acetylation and PERK activation facilitate eribulin-induced mitochondrial calcium accumulation and cell death. Article Snippet: These shRNA oligos were cloned into the pLKO.1blast vector (#26655; Addgene). .. For ATAT1 overexpression, the coding sequence (CDS) of ATAT1 was obtained by PCR using Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV‐GFP plasmid (Addgene, 25999) was used to express H2B‐GFP, and cells were selected by FACS (Beronja et al , ). eGFP‐αTAT1 was prepared from Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV-GFP plasmid (Addgene, 25999) was used to express H2B-GFP, and cells were selected by FACS (Beronja et al, 2010). eGFP-aTAT1 was prepared from Over Expression:Article Title: Microtubule acetylation and PERK activation facilitate eribulin-induced mitochondrial calcium accumulation and cell death. Article Snippet: These shRNA oligos were cloned into the pLKO.1blast vector (#26655; Addgene). .. For ATAT1 overexpression, the coding sequence (CDS) of ATAT1 was obtained by PCR using Sequencing:Article Title: Microtubule acetylation and PERK activation facilitate eribulin-induced mitochondrial calcium accumulation and cell death. Article Snippet: These shRNA oligos were cloned into the pLKO.1blast vector (#26655; Addgene). .. For ATAT1 overexpression, the coding sequence (CDS) of ATAT1 was obtained by PCR using Clone Assay:Article Title: Microtubule acetylation and PERK activation facilitate eribulin-induced mitochondrial calcium accumulation and cell death. Article Snippet: These shRNA oligos were cloned into the pLKO.1blast vector (#26655; Addgene). .. For ATAT1 overexpression, the coding sequence (CDS) of ATAT1 was obtained by PCR using Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV‐GFP plasmid (Addgene, 25999) was used to express H2B‐GFP, and cells were selected by FACS (Beronja et al , ). eGFP‐αTAT1 was prepared from Article Title: Distinct peroxisome populations differentially respond to alcohol-associated hepatic injury Article Snippet: Although peroxisomes are known to oxidize ethanol, metabolize lipids, and regulate oxidative stress, they remain understudied in the context of ethanol-induced liver injury.. We examined peroxisome early responses to alcohol-induced oxidative stress and lipid overload.. Analysis of peroxisomes labeled with catalase, an ethanol oxidizing enzyme, or ABCD3, a fatty acid transporter, revealed that distinct peroxisome populations differentially respond to ethanol. Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV-GFP plasmid (Addgene, 25999) was used to express H2B-GFP, and cells were selected by FACS (Beronja et al, 2010). eGFP-aTAT1 was prepared from Expressing:Article Title: Microtubule acetylation and PERK activation facilitate eribulin-induced mitochondrial calcium accumulation and cell death. Article Snippet: These shRNA oligos were cloned into the pLKO.1blast vector (#26655; Addgene). .. For ATAT1 overexpression, the coding sequence (CDS) of ATAT1 was obtained by PCR using Article Title: Distinct peroxisome populations differentially respond to alcohol-associated hepatic injury Article Snippet: Although peroxisomes are known to oxidize ethanol, metabolize lipids, and regulate oxidative stress, they remain understudied in the context of ethanol-induced liver injury.. We examined peroxisome early responses to alcohol-induced oxidative stress and lipid overload.. Analysis of peroxisomes labeled with catalase, an ethanol oxidizing enzyme, or ABCD3, a fatty acid transporter, revealed that distinct peroxisome populations differentially respond to ethanol. Article Title: Microtubule acetylation is a biomarker of cytoplasmic health during cellular senescence Article Snippet: The following secondary antibodies were used: DyLight T M 680 goat anti-mouse (35518, Invitrogen) and DyLight T M 800 goat anti–rabbit (SA5-10036, Invitrogen) for Western blotting and Alexa Fluor 488–, 555–, and 647–conjugated goat antibodies against rabbit and mouse IgG (Invitrogen) for immunofluorescence. .. The expression vectors used in this study were Article Title: Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation. Article Snippet: Article Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation Graphical abstract β α Ac Y β α Ac Y β α Ac Y β α Y β α Y β α AcetylationDetyrosination Hypoosmotic Cytoplasm density Isoosmotic Hyperosmotic VASH1-SVBP VASH1-SVBP VASH1-SVBP MAP7 MAP7 MAP7 αTAT1αTAT1 αTAT1 La tti ce e xp an si on La tti ce c om pa ct io n Plasmid Preparation:Article Title: Microtubule acetylation and PERK activation facilitate eribulin-induced mitochondrial calcium accumulation and cell death. Article Snippet: These shRNA oligos were cloned into the pLKO.1blast vector (#26655; Addgene). .. For ATAT1 overexpression, the coding sequence (CDS) of ATAT1 was obtained by PCR using Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV‐GFP plasmid (Addgene, 25999) was used to express H2B‐GFP, and cells were selected by FACS (Beronja et al , ). eGFP‐αTAT1 was prepared from Article Title: Distinct peroxisome populations differentially respond to alcohol-associated hepatic injury Article Snippet: Although peroxisomes are known to oxidize ethanol, metabolize lipids, and regulate oxidative stress, they remain understudied in the context of ethanol-induced liver injury.. We examined peroxisome early responses to alcohol-induced oxidative stress and lipid overload.. Analysis of peroxisomes labeled with catalase, an ethanol oxidizing enzyme, or ABCD3, a fatty acid transporter, revealed that distinct peroxisome populations differentially respond to ethanol. Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV-GFP plasmid (Addgene, 25999) was used to express H2B-GFP, and cells were selected by FACS (Beronja et al, 2010). eGFP-aTAT1 was prepared from Article Title: Microtubule acetylation is a biomarker of cytoplasmic health during cellular senescence Article Snippet: The following secondary antibodies were used: DyLight T M 680 goat anti-mouse (35518, Invitrogen) and DyLight T M 800 goat anti–rabbit (SA5-10036, Invitrogen) for Western blotting and Alexa Fluor 488–, 555–, and 647–conjugated goat antibodies against rabbit and mouse IgG (Invitrogen) for immunofluorescence. .. The expression vectors used in this study were Article Title: Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation. Article Snippet: Article Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation Graphical abstract β α Ac Y β α Ac Y β α Ac Y β α Y β α Y β α AcetylationDetyrosination Hypoosmotic Cytoplasm density Isoosmotic Hyperosmotic VASH1-SVBP VASH1-SVBP VASH1-SVBP MAP7 MAP7 MAP7 αTAT1αTAT1 αTAT1 La tti ce e xp an si on La tti ce c om pa ct io n FACS:Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV‐GFP plasmid (Addgene, 25999) was used to express H2B‐GFP, and cells were selected by FACS (Beronja et al , ). eGFP‐αTAT1 was prepared from Article Title: Centrosome amplification fine tunes tubulin acetylation to differentially control intracellular organization Article Snippet: .. The LV-GFP plasmid (Addgene, 25999) was used to express H2B-GFP, and cells were selected by FACS (Beronja et al, 2010). eGFP-aTAT1 was prepared from Recombinant:Article Title: Distinct peroxisome populations differentially respond to alcohol-associated hepatic injury Article Snippet: Although peroxisomes are known to oxidize ethanol, metabolize lipids, and regulate oxidative stress, they remain understudied in the context of ethanol-induced liver injury.. We examined peroxisome early responses to alcohol-induced oxidative stress and lipid overload.. Analysis of peroxisomes labeled with catalase, an ethanol oxidizing enzyme, or ABCD3, a fatty acid transporter, revealed that distinct peroxisome populations differentially respond to ethanol. Modification:Article Title: Microtubule acetylation is a biomarker of cytoplasmic health during cellular senescence Article Snippet: The following secondary antibodies were used: DyLight T M 680 goat anti-mouse (35518, Invitrogen) and DyLight T M 800 goat anti–rabbit (SA5-10036, Invitrogen) for Western blotting and Alexa Fluor 488–, 555–, and 647–conjugated goat antibodies against rabbit and mouse IgG (Invitrogen) for immunofluorescence. .. The expression vectors used in this study were Article Title: Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation. Article Snippet: Article Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation Graphical abstract β α Ac Y β α Ac Y β α Ac Y β α Y β α Y β α AcetylationDetyrosination Hypoosmotic Cytoplasm density Isoosmotic Hyperosmotic VASH1-SVBP VASH1-SVBP VASH1-SVBP MAP7 MAP7 MAP7 αTAT1αTAT1 αTAT1 La tti ce e xp an si on La tti ce c om pa ct io n Mutagenesis:Article Title: Microtubule acetylation is a biomarker of cytoplasmic health during cellular senescence Article Snippet: The following secondary antibodies were used: DyLight T M 680 goat anti-mouse (35518, Invitrogen) and DyLight T M 800 goat anti–rabbit (SA5-10036, Invitrogen) for Western blotting and Alexa Fluor 488–, 555–, and 647–conjugated goat antibodies against rabbit and mouse IgG (Invitrogen) for immunofluorescence. .. The expression vectors used in this study were Article Title: Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation. Article Snippet: Article Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation Graphical abstract β α Ac Y β α Ac Y β α Ac Y β α Y β α Y β α AcetylationDetyrosination Hypoosmotic Cytoplasm density Isoosmotic Hyperosmotic VASH1-SVBP VASH1-SVBP VASH1-SVBP MAP7 MAP7 MAP7 αTAT1αTAT1 αTAT1 La tti ce e xp an si on La tti ce c om pa ct io n |