Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. , B. Kaplan-Meier survival curves for wild-type and Usp54-deficient males (A) and females (B). C. TaqMan-based qRT-PCR analysis of Usp54 in MEFs and liver tissues from Usp54 +/+ and Usp54 KF/KF mice. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (**, P < 0.01). D. Body weight curves of Usp54 +/+ and Usp54 KF/KF female mice kept on standard diet. E. Body weight curves of Usp54 +/+ and Usp54 KF/KF female mice kept on high-fat diet and a representative image of females of each genotype at the end of the experiment. F. Total weight gain in the same animals. G. Percentage of gonadal and subscapular fat mass with respect to total body weight of the same animals. H. Mean adipocyte area in gonadal and skin fat. I. Representative histological images. Scale bar: 20 μm (gonadal fat) and 200 μm (skin fat). J. Average thickness of the subcutaneous fat deposits for each genotype. Statistical significance was assessed by a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. TaqMan-based qRT-PCR analysis of Usp54 expression in B16F10 cells transduced with the indicated Usp54-specific shRNA or the empty lentiviral vector (pLKO.1) as a control. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (***, P < 0.001). B. Number of metastases bigger than 200 μm in diameter. Statistical significance was assessed using a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; ***, P < 0.001). C. Representative images of lungs and histological analysis for each condition. Scale bar: 200 μm.

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Quantitative RT-PCR, Expressing, Transduction, shRNA, Plasmid Preparation, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: A The expression levels of Pcna, Casp3, Tnf , and Tgfb1 in cholangiocytes. Female C57BL/6 mice were fed DDC diet for 4 weeks with vehicle or 1,25(OH) 2 D 3 (5 µg/kg, 2x week) treatment and primary cholangiocytes were isolated ( n = 4/group). B Relative mRNA levels of Vdr . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks, and primary cholangiocytes, HSCs, liver macrophages, and LSECs were isolated ( n = 3/group). C Hepatic mRNA levels of Txnip . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks ( n = 3/group). D Relative mRNA levels of Txnip . Female C57BL/6 mice were treated with 1,25(OH) 2 D 3 (5 µg/kg, 2x week) for 4 weeks, and primary hepatocytes, cholangiocytes, HSCs, liver macrophages, and LSECs were isolated ( n = 3/group). E qPCR analysis. BMOL cells were pre-incubated with control or Vdr siRNA for 24 h and treated with 1,25(OH) 2 D 3 (10 µM) for 24 h ( n = 4 biological replicates). F Luciferase assay. BMOL cells were pre-incubated with control or Vdr siRNA for 24 h and treated with 1,25(OH) 2 D 3 (10 µM) for 6 h ( n = 4 biological replicates). Values represent means ± SDs. n.s., not significant. Significance determined by one-way ANOVA followed by Tukey’s multiple comparisons test ( A , E , F ) and unpaired two-tailed t test ( B , C , D ). Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Expressing, Isolation, Incubation, Control, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: Female Txnip fl/fl and Txnip ΔSox9 mice were fed DDC diet for 4 weeks. A Representative H&E staining ( n = 4/group). B IHC staining for pan-CK. Right: Numbers of pan-CK + cells ( n = 4/group). C Relative hepatic mRNA levels of Krt19 , Krt7 , and Sox9 ( Txnip fl/fl , n = 4; Txnip ΔSox9 , n = 3). D IF staining for F4/80 and Sirius-red staining. Right: The F4/80 + cell numbers and Sirius red-positive areas ( n = 4/group). Magnification: 200X. E , F qPCR analysis of genes involved in inflammation ( E ) and fibrosis ( F ) ( Txnip fl/fl , n = 4; Txnip ΔSox9 , n = 3). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Staining, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: Principal component analysis ( A ) and volcano map ( B ) for control and Txnip -overexpressing (OE) BMOL cells ( n = 3/group). C Top 20 enriched gene ontologies from DAVID analysis. D Heatmap of representative differentially expressed genes in control and Txnip OE BMOL cells. E Flow cytometric analysis of shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). F Effects of 1,25(OH) 2 D 3 on cell proliferation and apoptosis in shRNA control and shRNA Txnip BMOL cells, as assessed by Western blot analysis ( n = 3 biological replicates). G IF staining of pan-CK/BrdU and pan-CK/caspase-3 in liver tissues. Txnip fl/fl and Txnip ΔSox9 mice were DDC-fed for 4 weeks. Right: Percentages of pan-CK + BrdU + and pan-CK + caspase-3 + cells ( n = 4/group). Original magnifications: 400X and 800X. ( H ) qPCR analysis of cholangiocytes isolated from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Control, shRNA, Western Blot, Staining, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Vitamin D supplementation ameliorates ductular reaction, liver inflammation and fibrosis in mice by upregulating TXNIP in ductular cells

doi: 10.1038/s41467-025-59724-z

Figure Lengend Snippet: A Representative images of IF staining for F4/80/pan-CK and α-SMA/pan-CK in DDC-fed mice. F4/80 + liver macrophages and α-SMA + HSCs were observed near pan-CK + cells (arrows) near portal regions (1) and in parenchymal regions (2). Original magnifications: 400X and 800X. B qPCR analysis. Primary KCs isolated from C57BL/6 mice were co-cultured with shRNA control or shRNA Txnip BMOL cells for 24 h ( n = 4 biological replicates). C mRNA levels of cytokines in shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). D Relative mRNA expression levels of pro-inflammatory genes in cholangiocytes isolated from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). E Levels of TNF-α in culture media of shRNA control or shRNA Txnip BMOL cells treated with or without TNF-α neutralizing antibody (5 µg/ml) for 24 h ( n = 4 biological replicates). F Expression of pro-inflammatory genes in KCs isolated from C57BL/6 mice and co-cultured with shRNA control or shRNA Txnip BMOL cells plus TNF-α neutralizing antibody ( n = 4 biological replicates). G Relative mRNA expression levels of fibrosis-related genes. HSCs isolated from C57BL/6 mice were co-cultured with shRNA control or shRNA Txnip BMOL cells ( n = 3 biological replicates). H Relative mRNA expression levels of Tgfb1 in shRNA control and shRNA Txnip BMOL cells ( n = 3 biological replicates). I qPCR analysis of fibrosis-related genes in cholangiocytes from DDC-fed Txnip fl/fl and Txnip ΔSox9 mice ( n = 4/group). J Levels of TGF-β in culture media from shRNA control or shRNA Txnip BMOL cells treated with or without TGF-β neutralizing antibody (2 µg/ml) for 24 hours ( n = 3 biological replicates). ( K ) Expression of fibrosis-related genes in primary HSCs co-cultured with shRNA control or shRNA Txnip BMOL cells with or without TGF-β neutralizing antibody ( n = 3 biological replicates). Values represent means ± SDs. n.s., not significant. Significance determined by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: The corresponding cDNA was synthesized using a ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan).

Techniques: Staining, Isolation, Cell Culture, shRNA, Control, Expressing, Two Tailed Test

Journal: Theranostics

Article Title: MiR675-5p Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma

doi: 10.7150/thno.14700

Figure Lengend Snippet: Gain and loss of function suggests a critical role of miR675-5p on hypoxia modulation. (A) ELISA for HIF-1α nuclear level in U251 and HUVECs transfected with miR675-5p inhibitor and exposed to hypoxia for 6h. Data are expressed as ABS values at 450nm. ( B) Real-time PCR from U251 and HUVECs exposed to hypoxia after miR675-5p inhibitor or scramble control transfection. Data were normalized for β-actin and ΔΔct is expressed as fold of induction (FOI) of indicated genes after inhibitor transfection compared to scramble control (dashed line). (C) ELISA assay for VEGF level in supernatant from both cell lines transfected with miRNA inhibitor or negative control and exposed to hypoxia for 6h. Data are expressed as pg/ml of soluble VEGF. Values are presented as the mean ± SD. Inhibitor vs scramble * p<0.05; **p<0.0; ***p<0.001. (D) HIF-1α nuclear level analyzed by ELISA assay in U251 and HUVEC cells after 18h of miR675-5p mimic transfection in normoxia. Data are expressed as ABS values at 450nm. (E) Real-time PCR performed on U251 and HUVEC cells after miR675-5p mimic-transfection. Data were normalized for β-actin, and ΔΔct is expressed as fold of induction (FOI) in cells transfected with mimic compared to control (dashed line). (F) ELISA assay for VEGF level in supernatant from both cell lines 18h after mimic transfection. Data are expressed as pg/ml of soluble VEGF. Values are presented as the mean ± SD. Mimic vs scramble * p<0.05; **p<0.01;***p<0.001.

Article Snippet: To make the mixture stable, the oligomers were injected by using a kit for delivering miRNA into animal tissue by systemic administration (AteloGene in vivo siRNA/miRNA Transfection kit, CosmoBio, DBA, Italy).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction, Negative Control

Journal: Theranostics

Article Title: MiR675-5p Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma

doi: 10.7150/thno.14700

Figure Lengend Snippet: miR675-5p in vivo is able to enhance hypoxia establishment and neo-angiogenesis. (A) Luciferase in vitro assay performed on U251-HRE lysated cells after 18h of miR675-5p mimic-transfection. Data are represented as RLU (luminescent counts normalized to protein content) of mimic-treated cells compared to scramble-treated cells. Mimic vs scramble ***p<0.001. (B) Left panel: Representative (n=5) 2D bioluminescent (rainbow scale) of U251-HRE-mCherry tumours in scramble and miR675-5p mimic-treated mice (one of 5 mice) at day 12 (pre-treatment), day 15 (after two doses) and day 19 (end of treatment). Images are presented with the same scale bar . Right panel: Graphical representation of Luciferase activity over time during treatment. Data are presented as average radiance (photons/s/cm^2/steradian). (C) Left panel: Representative 2D images of mice co-injected with HypoxiSense 680 (BlueHot scale) and IntegriSense 750 (YellowHot scale) and axial images of IntegriSense750 co-registered with CT scan. All scans were performed at the final time point (day 19). Right panel: Quantification of HypoxiSense680 and IntegriSense750 fluorescence by ROI analysis for scramble and mimic-treated mice. Data are expressed as average radiance efficiency [(photons/s/cm^2/steradian)/(μW/cm^2)]. (D) Left panel: Representative (n=5) 2D mCherry fluorescent signal (rainbow scale) of U251-HRE-mCherry tumours in scramble and miR675-5p mimic-treated mice at day 19. Right panel: Graphical representation of mCherry activity at the end of treatment. Data are presented as average radiance efficiency [(photons/s/cm^2/steradian)/(μW/cm^2). Images are presented with the same scale bar. Mimic-treated mice vs scramble- treated mice * p<0.05; **p<0.01; ***p<0.001.

Article Snippet: To make the mixture stable, the oligomers were injected by using a kit for delivering miRNA into animal tissue by systemic administration (AteloGene in vivo siRNA/miRNA Transfection kit, CosmoBio, DBA, Italy).

Techniques: In Vivo, Luciferase, In Vitro, Transfection, Activity Assay, Injection, Computed Tomography, Fluorescence

Journal: Theranostics

Article Title: MiR675-5p Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma

doi: 10.7150/thno.14700

Figure Lengend Snippet: miR675-5p regulates HIF-1α mRNA stability by VHL/HuR axis. (A) Real-time PCR for VHL was done on U251 cells 18h after transfection with 5pM, 15pM, 30pM and 60pM of miR675-5p mimic. The dashed line indicates the control sample (scramble-treated). Values are presented as the mean ± SD. Mimic vs scramble * p<0.05; **p<0.01 . (B) Western Blot for VHL and β-actin from U251 cells transfected with miR675-5p mimic (5pM) or scramble negative control. (C) Expression of VHL-regulated miRNA was validated by Luminescence assay kit, a secondary reporter was used for transfection normalization. Data are expressed as Relative Luciferase Activity in pGL3-pVHL-WT construct transfected with miR675-5p or miR21-5p and their relative scrambles. pGL3-control is represented as dashed line. The mean ± SD of three independent experiment is shown, and each sample was assayed in triplicate. (D) Left panel: Real-time PCR for HIF-1α performed on U251 and HUVEC cells 6 hours post-hypoxia. Data were normalized for β-actin, and ΔΔCt is expressed as fold of induction (FOI) of HIF-1α in hypoxia vs normoxia. Right panel: Real-time PCR performed on U251 and HUVEC cells 6 hours post-hypoxia and after miRNA675-5p inhibitor transfection. Data were normalized for β-actin, and ΔΔCt is expressed as fold of induction (FOI) of HIF-1α in hypoxia in inhibitor vs normoxia. (E) Left panel: Western Blot for HuR and β-actin from U251 cells transfected with miR675-5p inhibitor (5pM) or scramble negative control after 6h of hypoxia. Right panel: RNA-immunoprecipitation for HuR. Real time PCR for VEGF and HIF-1α were performed starting from HuR co-immunoprecipitated RNA in different culture conditions. Ct value was normalized to the Input RNA Ct value. Data are expressed as fold enrichment above the input percentage and presented as the mean ± SD. Inhibitor vs scramble negative control ***p<0.001. (F) Molecular mechanism of miRNA 675-5p in hypoxia condition. The dashed line indicates our experimental results.

Article Snippet: To make the mixture stable, the oligomers were injected by using a kit for delivering miRNA into animal tissue by systemic administration (AteloGene in vivo siRNA/miRNA Transfection kit, CosmoBio, DBA, Italy).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Western Blot, Negative Control, Expressing, Luminescence Assay, Luciferase, Activity Assay, Construct, Immunoprecipitation

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: PRIMERS USED TO GENERATE SMALL INTERFERING RNA FROM HUMAN AIRWAY SMOOTH MUSCLE CELL cDNA

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Small Interfering RNA

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Analysis of the effect of siRNA-induced suppression of integrin α7 mRNA and protein over 6 days of serum-free culture. (A) PCR analysis; α7 PCR product size = 599 base pairs (bp). (B) Western blotting for integrin α7B. Also shown is Western blot analysis of the effects of α7 siRNA on desmin and smooth muscle α-actin accumulation after 6 days of serum deprivation. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; †P < 0.05, compared with control cultures (6-day serum-free media exposed to transfection reagent without siRNA).

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Control, Transfection

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Western blot analysis showing the effect of siRNA silencing of integrin α7 on the accumulation of integrins α3A (A), α6A (B), α5 (C), and β1 (D) over 6 days of serum deprivation. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; †P < 0.05; ns = not significant compared with control cultures (6-day serum free media exposed to transfection reagent without siRNA). Panels E–H show phase contrast images of human ASM cells in the absence (transfection reagent alone; E) or presence of siRNA directed at integrin α3 (F), α6 (G), or α7 (H). Bar = 50 μm.

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Control, Transfection

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Western blot analysis showing confirmation of effect of siRNA for integrin α3 (A) or α6 (B) on protein abundance of integrin α3A and α6A. Grouped data, which are expressed as relative units to Day 0, represent results from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; †P < 0.05; ns = not significant compared with control cultures (6 days of serum-free media exposed to transfection reagent without siRNA).

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Quantitative Proteomics, Control, Transfection

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: EFFECTS OF TREATMENT WITH α3 OR α6 INTEGRIN siRNA ON ABUNDANCE OF α5, AND α7B PROTEIN IN 6-DAY SERUM DEPRIVED HUMAN AIRWAY SMOOTH MUSCLE CELL CULTURES

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Quantitative Proteomics

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Western blot analysis showing the effect of siRNA silencing of integrin α3 (A) or integrin α6 (B) on the accumulation of desmin and smooth muscle α-actin over 6 days of serum free culture. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; ns = not significant compared with control cultures (6 days of serum-free media exposed to transfection reagent without siRNA).

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Control, Transfection

Journal: Cell reports

Article Title: CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition

doi: 10.1016/j.celrep.2021.108768

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit , Vector Labs , MP-7401.

Techniques: Western Blot, Immunohistochemistry, Virus, Recombinant, Protease Inhibitor, Reverse Transcription, SYBR Green Assay, Flow Cytometry, Luminescence Assay, Luciferase, Polymer, Plasmid Preparation, Expressing, Chromatin Immunoprecipitation, shRNA, Software

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression in Breast Cancer PDXs That Have Acquired Resistance to PI3Kα Inhibition (A) Relapse of an HCI-011 PDX after treatment with GDC-0032 (HCI-011R). (B) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts receiving drug vehicle (n = 5) or GDC-0032 (n = 8). p values were defined using two-sided Wald t tests. (C) Immunoblots of lysates from (B) and from an HCI-011 tumor. (D) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts treated with drug vehicle (n = 2), drug vehicle plus tamoxifen (n = 3), and tamoxifen plus GDC-0032 (n = 5). p values were defined using two-sided Wald t tests. (E) Immunoblots of lysates from (D). Mean ± standard deviation (n = 2 to 4). p values were calculated using two-sided Welch's t tests. (F) Gene expression (Log2) enrichment (heatmap: red, positive; white, neutral; blue, negative) of FOXM1 gene targets in HCI-011 (n = 4) and in HCI-011R xenografts (n = 8). The top row shows false discovery rate (FDR)-adjusted p values. (G) Gene expression (Log2) enrichment of genes mediating resistance to BYL-719 in HCI-011 (n = 4 for vehicle and drug) and in HCI-011R (n = 5 vehicle, n = 8 GDC-0032). FDR-adjusted p values of drug-resistant versus drug-sensitive differential expression are shown in the top row. (H) Mean tumor volume (cm 3 ) ± SEM of drug-resistant T47DR tumors after long-term treatment with GDC-0032. p values were defined using two-sided Wald t tests. (I) Immunoblot of tumors lysates from (H). See also Figure S5 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Gene Expression, Quantitative Proteomics

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression Contributes to Drug Resistance and Can Be Explained by Cytoplasmic Localization of FOXO3a (A) Representative confocal microscopy images of T47D PTEN wt (Ctrl) and PTEN KO cells drug-treated for 72 h. FOXO-3a staining (green), DAPI staining (blue). (B) Relative number of red fluorescent MCF7 and T47D cells (mStrawberry-positive cells, co-expressing FOXM1C) in mixtures with parental controls following treatment for 120 h. p values were calculated using two-sided Welch's t tests. (C) Viability of HCI-011R PDTCs treated with the indicated drugs for 120 h. Left: viability of HCI-011R PDTCs treated for 120 h. Mean ± standard deviation (five technical replicates, n = 3 experiments). Right: mean ± standard deviation (five technical replicates, n = 4 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (D) Viability of cells dissociated from a T47DR tumor treated with the indicated drugs for 120 h. Left: mean ± standard deviation (five technical replicates, n = 4 experiments). Right: mean ± standard deviation (five technical replicates, n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (E) Viability of T47D PTEN KO cells expressing FOXM1 targeting sequences (shFOXM1 [sequence 1 or 2]) or a non-targeting control sequence (shCtrl) treated for 120 h. Mean ± standard deviation (n = 5, five technical replicates). p values were calculated using two-sided Welch's t tests on the averages of replicates. (F) Viability of HCI-011R PDTCs expressing doxycycline-inducible shRNA sequences targeting FOXM1 (sequence 1) or a control shRNA and treated for 120 h. Mean ± standard deviation (three technical replicates). p values were calculated using two-sided Welch's t tests on the averages of the replicates. (G) LDH activity in the cells used in (E) and treated for 96 h. Figure shows the mean ± standard deviation (n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (H) Immunoblots of lysates from cells used in (G). (I) Mean tumor volume (cm 3 ) ± SEM of T47D PTEN KO xenografts expressing doxycycline-inducible shFOXM1 (sequence 1) or shCtrl, following prolonged GDC-0032 treatment. One cohort received standard food (shCtrl n = 3 and shFOXM1 n = 3) and the other cohort food plus doxycycline (shCtrl n = 3 and shFOXM1 n = 4; 0.2 g/kg food pellet Harlan D.98186). p values were defined using two-sided Wald t tests. (J) Immunoblot of lysates from tumors in (I). p values were calculated using two-sided Welch's t tests. See also Figure S6 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Confocal Microscopy, Staining, Standard Deviation, Sequencing, Control, shRNA, Activity Assay, Western Blot