Journal: Emerging Infectious Diseases

Article Title: Identifying Influenza Viruses with Resequencing Microarrays

doi: 10.3201/eid1204.051441

Figure Lengend Snippet: Hybridization images of the respiratory pathogen microarray (RPM) version 1 prototype regions for 3 influenza virus isolates and trivalent FluMist vaccine. A) A/H1N1, B) A/H3N2, C) influenza B, and D) trivalent FluMist vaccine. In A, B, and C, only the influenza-specific tiled prototype regions of RPM version 1 are shown. Hybridization-positive identifications are shown on the right. In D, the image of the entire RPM version when hybridized with FluMist vaccine is shown. The single influenza prototype region that was hybridization negative is denoted on the right. E) Magnification of a portion of profile B showing an example of the primary sequence data generated by the hybridization of randomly amplified targets to the RPM version 1 HA3 probe set. The primary sequence generated can be read from left to right. HA, hemagglutinin; NA, neuraminidase; IQEX, internal positive hybridization control (Affymetrix); M, matrix.

Article Snippet: After the prehybridization step, 167.5 μL of hybridization cocktail master mix (3 mol/L tetramethylammonium chloride, 10 mmol/L Tris, pH 7.8, 0.01% Tween 20, 0.5 mg/mL bovine serum albumin, 0.1 mg/mL herring sperm DNA [Promega, Madison, WI, USA], 50 pmol/L Oligo B2 [Affymetrix Inc.]) and biotin-labeled DNA fragments were heated for 5 min at 95°C, equilibrated for 5 min at 45°C, and added to RPM version 1.

Techniques: Hybridization, Microarray, Virus, Sequencing, Generated, Amplification, Control

Journal: Analytical Chemistry

Article Title: Optimized Light-Directed Synthesis of Aptamer Microarrays

doi: 10.1021/ac400746j

Figure Lengend Snippet: Figure 3. Normalized streptavidin binding signal for a St-2-1 aptamer sequence synthesized with a photodeprotection light exposure gradient between 0.2 and 18 J/cm2. The black squares represent data from a microarray synthesized with a maximum surface density of oligonucleotides. The red circles are from an equivalent experiment, but with a microarray with an oligonucleotide surface density reduced by 50%.

Article Snippet: After blocking, the arrays were incubated with streptavidin (ProSpec, USA) using an incubation mixture containing 1xPBS/1 mM MgCl2/0.01% Tween-20/1% BSA/10 μg per mL streptavidin (PBS, Ambion, USA; MgCl2, Sigma, Germany; BSA, Sigma, USA).

Techniques: Binding Assay, Sequencing, Synthesized, Microarray

Journal: Analytical Chemistry

Article Title: Optimized Light-Directed Synthesis of Aptamer Microarrays

doi: 10.1021/ac400746j

Figure Lengend Snippet: Figure 5. Aptamer binding and hybridization signal comparison between microarrays synthesized with four different surface chem- istries (left to right): Corning UltraGAPS/amino-modified; Schott E/ epoxy-modified, ring-opened; Schott A+/amino-modified; and in- house hydroxyl-functionalized. Top: St-2-1 aptamer−streptavidin binding signal. Bottom: hybridization signal for three probes hybridized with 1 and 100 pM complementary sequences. Error bars are the standard deviation among replicates.

Article Snippet: After blocking, the arrays were incubated with streptavidin (ProSpec, USA) using an incubation mixture containing 1xPBS/1 mM MgCl2/0.01% Tween-20/1% BSA/10 μg per mL streptavidin (PBS, Ambion, USA; MgCl2, Sigma, Germany; BSA, Sigma, USA).

Techniques: Binding Assay, Hybridization, Comparison, Synthesized, Standard Deviation

Journal: Analytical Chemistry

Article Title: Optimized Light-Directed Synthesis of Aptamer Microarrays

doi: 10.1021/ac400746j

Figure Lengend Snippet: Figure 4. Fluorescent images of sections of microarrays synthesized on different substrates. (A) Corning UltraGAPS, (B) Schott Epoxy ring- opened, (C) Schott A+, (D) Schott Glass D hydroxyl-functionalized with N-(3-triethoxysilylpropyl)-4-hydroxybutryramide. Green features are hybridization signals from Cy3-labeled sequences. Red features are from Cy5-labeled biotin binding to the streptavidin−aptamer pairs. A scheme identifying the sequences corresponding to the spots can be found in the Supporting Information. The arrays were synthesized with light exposures of 11 J/cm2 and with 32 × 32 μm features (4 DMD mirrors) separated by gaps of 48 μm. All images were acquired with the same scanner settings.

Article Snippet: After blocking, the arrays were incubated with streptavidin (ProSpec, USA) using an incubation mixture containing 1xPBS/1 mM MgCl2/0.01% Tween-20/1% BSA/10 μg per mL streptavidin (PBS, Ambion, USA; MgCl2, Sigma, Germany; BSA, Sigma, USA).

Techniques: Synthesized, Hybridization, Labeling, Binding Assay

Journal: Analytical Chemistry

Article Title: Optimized Light-Directed Synthesis of Aptamer Microarrays

doi: 10.1021/ac400746j

Figure Lengend Snippet: Figure 7. Effect of mutations in the 29-mer St-2-1 aptamer sequence on the binding affinity. Binding was tested using an on-array streptavidin binding assay. Black bar, St-2-1; blue bars, mutation in the terminal stem; green bars, mutation in the bulges; red bars, mutation in the sequences between the bulges. Error bars are based on 12 replicates on the array. Signal negative control St-2-1_rev =28.4.

Article Snippet: After blocking, the arrays were incubated with streptavidin (ProSpec, USA) using an incubation mixture containing 1xPBS/1 mM MgCl2/0.01% Tween-20/1% BSA/10 μg per mL streptavidin (PBS, Ambion, USA; MgCl2, Sigma, Germany; BSA, Sigma, USA).

Techniques: Sequencing, Binding Assay, Mutagenesis, Negative Control

Journal: Analytical Chemistry

Article Title: Optimized Light-Directed Synthesis of Aptamer Microarrays

doi: 10.1021/ac400746j

Figure Lengend Snippet: Figure 8. Effect of base pair replacements in the double-stranded parts of St-2-1. Binding was tested using an on-array streptavidin binding assay. Black bar, St-2-1; blue bars, mutation in the terminal stem; red bars, mutation in the sequence between the bulges. Error bars are based on 12 replicates on the array. Secondary structure of the variants A12T23, G12C23, and C12G23. The structural variant C12G23 correlates with reduced streptavidin binding on-array.

Article Snippet: After blocking, the arrays were incubated with streptavidin (ProSpec, USA) using an incubation mixture containing 1xPBS/1 mM MgCl2/0.01% Tween-20/1% BSA/10 μg per mL streptavidin (PBS, Ambion, USA; MgCl2, Sigma, Germany; BSA, Sigma, USA).

Techniques: Binding Assay, Mutagenesis, Sequencing, Variant Assay

Journal: Applied and Environmental Microbiology

Article Title: Discovery of Stable and Variable Differences in the Mycobacterium avium subsp. paratuberculosis Type I, II, and III Genomes by Pan-Genome Microarray Analysis

doi: 10.1128/aem.01683-08

Figure Lengend Snippet: FIG. 1. Microarray data for validation hybridization comparing the two sequenced reference strains, using M. avium subsp. hominissuis 104 as the test strain and M. avium subsp. paratuberculosis K-10 as the reference strain per other strain comparisons. Scatter plots show the signal intensities for the test (y axis) versus the reference (x axis) strain channels. Data points are colored according to the BLAST prediction based on the sequence: black, predicted to be present in both the test and reference strains; light gray, predicted to be present in the test strain only; dark gray, predicted to be present in the reference strain only. Diagonal lines represent twofold cutoffs applied to each strain for each of the analysis methods used. MAH, M. avium subsp. hominissuis; MAP, M. avium subsp. paratuberculosis.

Article Snippet: The labeled sample was loaded on to a prehybridized (3.5 SSC [1 SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% SDS, 10 mg/ml bovine serum albumin) microarray under two 22- by 22-mm LifterSlips (Erie Scientific), sealed in a humidified hybridization cassette (Corning), and hybridized overnight by immersion in a water bath at 55°C for 16 to 20 h. Slides were washed once in 400 ml 1 SSC, 0.06% SDS at 55°C for 2 min and twice in 400 ml 0.06 SSC for 2 min. Microarray data analysis.

Techniques: Microarray, Biomarker Discovery, Hybridization, Sequencing

Journal: Oncology Reports

Article Title: HOXA11 plays critical roles in disease progression and response to cytarabine in AML

doi: 10.3892/or.2021.8101

Figure Lengend Snippet: Expression levels of Hox clusters A, B, C, D and Meis1 genes in paired MLL/AF10 leukemia cells harboring wild-type or oncogenic RAS pathway mutations. (A-C) Heat maps of genes based on cDNA microarray data between paired cell lines: (A) 12G and AK3G, (B) AKw1G and AK3G, and (C) APw-1 and APm-1. Raw values are log2-transformed. Red and black indicate high and low levels of gene expression, respectively. Color bar depicts log2-transformed value of genes. (D) Reverse transcription-quantitative PCR analysis was performed to determine the level of Hoxa11 expression in 12G, AKw1G, AK3G, AK2G, APw-1 and APm-1 cells. Assays were performed in triplicate and data are representative of three independent experiments. Error bars indicate SD. (E) Western blotting was performed to determine the protein level of Hoxa11 in cells of the different cell lines. The detection of β-actin and Gapdh served as a loading control for immunoblot analysis. MLL, lysine methyltransferase 2A; 12G, cells with MLL/AF10(OM-LZ) alone; AK3G, cells with MLL/AF10(OM-LZ) and oncogenic KRAS G12C; AKw1G, cells with MLL/AF10(OM-LZ) and wild-type KRAS ; APw-1, cells with MLL/AF10(OM-LZ) and wild-type PTPN11 ; APm-1, cells with MLL/AF10(OM-LZ) and oncogenic PTPN11 G503A; PTPN11 , tyrosine-protein phosphatase non-receptor type 11.

Article Snippet: The membrane was blocked in 5% bovine serum albumin at 4°C (Sigma-Aldrich; Merck KGaA) for 1 h, and then incubated with primary antibodies at 4°C overnight against mouse Hoxa11 (1:5,000; cat. no. NBP1-80228; Novus Biologicals, Ltd.), β-actin or Gapdh (1:10,000; cat. nos. sc-47778 or sc-32233; Santa Cruz Biotechnology, Inc.), followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; cat. nos.

Techniques: Expressing, Microarray, Transformation Assay, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Molecular Cancer Therapeutics

Article Title: Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells bothin vitroandin vivo

doi: 10.1158/1535-7163.mct-05-0178

Figure Lengend Snippet: Figure 1. Immunohistochemical staining of clusterin in a breast cancer tissue microarray. Tissue microarrays were immunostained for clusterin and scored by two pathologists. Specimens were graded from 0 to +3 intensity representing the range from no staining to heavy staining. Three representative images at 40 magnification show high (A), moderate (B), and low (C) clusterin staining.

Article Snippet: The slides were successively transferred to 3% H2O2 for 10 minutes and then incubated overnight with goat anti-human clusterin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at the concentration of 1:600 in 1% bovine serum albumin.

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Molecular Cancer Therapeutics

Article Title: Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells bothin vitroandin vivo

doi: 10.1158/1535-7163.mct-05-0178

Figure Lengend Snippet: Figure 4. Clusterin increases in MCF-7 cells treated with paclitaxel or estrogen withdrawal. To deter- mine whether estrogen withdrawal or paclitaxel treatment affects clus- terin expression, cells with treated with either estrogen withdrawal in charcoal-stripped serum (CSS) or 100 nmol/L paclitaxel. After 2 d of treatment, cells were treated with 50 nmol/L OGX-011 for 2 d. Protein was harvested 48 h after the final treat- ment of OGX-011. Three different isoforms of clusterin were found: 55- kDa nuclear clusterin and the 60- and 45-kDa bands representing the two heterodimers of full-length secreted clusterin.

Article Snippet: The slides were successively transferred to 3% H2O2 for 10 minutes and then incubated overnight with goat anti-human clusterin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at the concentration of 1:600 in 1% bovine serum albumin.

Techniques: Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells bothin vitroandin vivo

doi: 10.1158/1535-7163.mct-05-0178

Figure Lengend Snippet: Figure 7. OGX-011 suppresses clusterin levels in MCF-7 xenografts in vivo. MCF-7 xenografts were harvested at day 42 and Western blot analysis was done to quantify clusterin levels. Loading levels were normalized by blotting with (1:2,000) anti-vinculin and densitometric analysis. Two isoforms of clusterin were found: nuclear clusterin (n-CLU) and full-length secreted clusterin (s-CLU). Three representative samples are shown in each group.

Article Snippet: The slides were successively transferred to 3% H2O2 for 10 minutes and then incubated overnight with goat anti-human clusterin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at the concentration of 1:600 in 1% bovine serum albumin.

Techniques: In Vivo, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells bothin vitroandin vivo

doi: 10.1158/1535-7163.mct-05-0178

Figure Lengend Snippet: Figure 8. Immunostaining of MCF-7 xenografts treated with paclitaxel + OGX-011. Tissue microarrays spotted with MCF-7 tumors were immunostained with primary goat anti-human clusterin antibody. The staining intensity of malignant tissue was graded from 0 to +3 intensity representing the range from no staining to heavy staining. A, represen- tative images from OGX-011 + paclitaxel or control oligonucleotide + paclitaxel (200). B, mean scores from the five samples.

Article Snippet: The slides were successively transferred to 3% H2O2 for 10 minutes and then incubated overnight with goat anti-human clusterin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at the concentration of 1:600 in 1% bovine serum albumin.

Techniques: Immunostaining, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: CCN1 expression in Panc1 is increased by co-culturing with mPSCs. ( A , B ) The expression level of CCN1/Ccn1 in Panc1-WT ( A ) and mPSCs ( B ) from monospheroids and heterospheroids on day 5 was analyzed via RNA-seq and RT-PCR. Represented are RPKM (Reads Per Kilobase per Million mapped reads) values from the published expression profiling , and the mRNA fold changes presented here were normalized to Panc1 or mPSCs monospheroids. All data shown are means with SEM from three independent biological replicates. ( C ) The representative Western blot illustrates the expression of CCN1 protein levels in Panc1-WT and 3 different Panc1-CCN1-KO clones (C3, C7, and F3) cultured in 2D. ( D ) Representative pictures of Panc1-WT and Panc1-CCN1-KO clones C3, C7, and F3 monospheroids. Scale bars represent 500 μm. M, monospheroid; H, heterospheroid. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Clone Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: CCN1 negatively regulates stemness and positively affects gemcitabine transporter and activating genes. ( A ) The mRNA expression of the stemness marker gene CD24, the epithelial marker genes CK19 and CDH1, matricellular protein CTGF/CCN2, SHH signaling mediator GLI1, proliferation marker MKI67, gemcitabine transporter SLC29A1, and gemcitabine-activating enzyme DCK in Panc1-WT and Panc1-CCN1-KO clones (C3, C7, and F3) from monospheroids were analyzed with qRT-PCR. The mRNA fold changes were normalized to Panc1-WT monospheroids. All data shown are means with SEM from three independent biological replicates. ( B ) The protein level of DCK in Panc1-WT and Panc1-CCN1-KO clones (C3, C7, and F3) from 2D culture was analyzed with Western blot (WB). The relative ratio of DCK was quantified via DCK amount per lane/Tubulin amount per lane and then normalized to Panc1-WT. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. n = 4, M = protein weight marker. ( C ) The associations between the expression of these marker genes and CCN1 were fitted using linear regression based on the microarray dataset GSE71729. n = 145 patient samples. ( D ) Protein levels of CCN1, DCK (yellow arrow), and Tubulin for Panc1 WT, Panc1-CCN1-KO C7, and the CCN1-rescued Panc1-CCN1-KO C7 carried out with adenoviral infection. The control for adenoviral infection was carried out with Panc1-CCN1-KO C7 cells infected with adenovirus containing an eGFP expression cassette.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, Marker, Clone Assay, Quantitative RT-PCR, Western Blot, Microarray, Infection, Control

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: CCN1 increases chemotherapy sensitivity for gemcitabine but not paclitaxel and SN38. ( A ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3 and C7) cells grown in 2D culture after 72 h treatment with different doses of gemcitabine were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC (negative control). ( B ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3 and C7) cells in monospheroids after 72 h treatment with different concentrations of gemcitabine were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC. ( C ) The ratios of epithelial specific caspase-cleaved cytokeratin 18 (ccCK18) for Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from monospheroids and heterospheroids after 72 h treatment with 50 µM gemcitabine were measured via M30 Apoptosense ® CK18 kit. ( D ) The relative ratios of the epithelial-specific cleaved ccCK18 following 72 h gemcitabine treatment of Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from heterospheroid versus monospheroid culture are shown. ( E ) The mRNA expression of Ccn1 in mPSCs from heterospheroids co-cultured with either Panc1-WT or Panc1-CCN1-KO (C3, C7, and F3) cells is depicted. The mRNA fold changes were normalized to mPSCs co-cultured with Panc1 WT. ( F ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from monospheroids after 72 h treatment with paclitaxel [1 μM] were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC. ( G ) The relative cell viability of Panc1-WT and Panc1-CCN1-KO (C3, C7, and F3) cells from monospheroids after 72 h treatment with SN38 [1 μM] were measured via CellTiter-Glo ® 3D Cell Viability Assay and normalized to NC. All data shown are means with SEM of at least three independent biological replicates. *, p < 0.05; **, p < 0.01. M, monospheroid; H, heterospheroid; NC, negative control; GEM, gemcitabine; PAC, paclitaxel, SN38, active form of irinotecan.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Viability Assay, Negative Control, Expressing, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: LPA and TGFB1 upregulate CCN1 expression in Panc1. ( A ) Analyses of mRNA expression of selected genes in Panc1-WT monospheroids cultured in low-serum condition following stimulation with 20 μM LPA or 5 μg/mL recombinant TGFB1 for 72 h. ( B ) Analyses of mRNA expression of selected genes in Panc1-WT monospheroids cultured in high-serum condition following stimulation with 5 μg/mL recombinant TGFB1 for 72 h. ( C ) The mRNA expressions of CCN1, DCK, and SCL29A1 in Panc1 cells from heterospheroids cultured in high-serum condition stimulated with 5 ng/mL TGFB receptor kinase inhibitor, or 10 μM ATX inhibitor PF8380, or a combination of both inhibitors for 72 h are depicted. The mRNA fold changes presented were normalized NC. All data shown are means with SEM of three biological replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001. ( D ) The associations between ENPP2, TGFB1, and CYR61(CCN1) were fitted using linear regression based on the microarray dataset GSE71729. n = 145 patient samples. ATX, autotaxin; INH, inhibitor.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, Cell Culture, Recombinant, Microarray

Journal: International Journal of Molecular Sciences

Article Title: The Crosstalk Analysis between mPSCs and Panc1 Cells Identifies CCN1 as a Positive Regulator of Gemcitabine Sensitivity in Pancreatic Cancer Cells

doi: 10.3390/ijms25179369

Figure Lengend Snippet: TGFB1 and LPA positively regulate Ccn1 in mPSCs and shift mPSCs to the myCAF subtype. ( A ) Analyses of mRNA expression of genes in mPSCs monospheroids stimulated with 5 μg/mL recombinant TGFB1 for 72 h. ( B ) Analyses of mRNA expression of genes in mPSCs monospheroids stimulated with 20 μM LPA for 72 h. The fold changes in the expression of selected genes were normalized to NC. ( C ) Analysis of the mRNA expression of genes in mPSCs from heterospheroid co-cultured with Panc1-WT cells stimulated with 10 μM ATX/ Enpp2 inhibitor PF8380 on day 5 for 72 h is depicted. The mRNA fold changes were normalized to NC. All data shown are means with SEM of three biological replicates. ( D ) The associations between ACTA2 and ENPP2 using linear regression based on the microarray dataset GSE71729. n = 145 patient samples. NC, negative control. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Membranes were blocked for 2 h in 1X Tris-buffered saline with 0.1% Tween 20 buffer (TBS-T, ThermoFisher, #28360) containing 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA, A7906) and then incubated with primary antibodies (pAB): CCN1 (Boster Bio, PB9549, 1:1000), DCK (LifeSpan BioSciences, Lynnwood, WA, USA, LS-B10837, 1:1000), and Tubulin (Abcam, Cambridge, UK, ab7291, 1:1000) overnight or for 2 days at 4 °C.

Techniques: Expressing, Recombinant, Cell Culture, Microarray, Negative Control