Journal: JCI Insight

Article Title: PLA2G4A promotes right-sided colorectal cancer progression by inducing CD39+γδ Treg polarization

doi: 10.1172/jci.insight.148028

Figure Lengend Snippet: CT26- Pla2g4a and CT26-Vec cells were cocultured with intraepithelial lymphocytes derived from the ileum (IL-IELs), colon (CO-IELs), or spleen of BALB/c mice. (A and C ) Representative pictures showed the number of CT26 cells in the coculture system after 12 ( A ) and 36 hours ( C ). Scale bars: 200 μm. ( B and D ) Bar diagrams summarizes the cell numbers of CT26- Pla2g4a and CT26-Vec cells cocultured with IL-IELs after 12 ( B ) and 36 ( D ) hours. Data are shown as mean ± SEM, using 2-way ANOVA followed by Sidak’s multiple-comparisons test. ( E–G ) Representative histogram plot and bar diagram summarizes the expression of CD39 on γδ T cells in IL-IELs ( E ), CO-IELs ( F ) and splenocytes ( G ) group. ( H ) Representative flow cytometric analysis of CD39 + Tregs in the total CD3 + T cells from splenocytes. Bar diagram summarizes the percentages of CD39 + γδ Tregs. Unpaired 2-tailed t test was used to compare variables. ( I ) Silencing Pla2g4a in CT26- Pla2g4a rescued the antitumor response mediated by γδ T cells. CT26- Pla2g4a were transfected with Pla2g4a siRNA or control siRNA. After 24 hours, CT26-Vec, CT26- Pla2g4a , and CT26- Pla2g4a –knock-down cells were cocultured with γδ T cells sorted from BALB/c mice with or without anti-PLA2G4A antibody (5 mg/mL). Scale bars: 200 μm. ( J ) Bar diagram summarizes the cell number of CT26. ( K ) Representative histogram plot of CD39 expression on γδ T cells in each group by flow cytometry. Bar diagram summarizes the frequency of CD39 + γδ Tregs/γδ T cells. One-way ANOVA followed by Tukey’s multiple-comparison test was used for multiple-group analysis. All of the experiments were repeated 3 times; n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For rescue experiments, γδ T cells sorted from IELs of BALB/c mice were cocultured with CT26-Vec or CT26- Pla2g4a (γδ T/CT26 = 1:1) with siRNA pretreatment for 2 hours using a mouse TCR γ/δ T cell Isolation Kit (catalog 130-092-125; Miltenyi Biotec).

Techniques: Derivative Assay, Expressing, Transfection, Control, Knockdown, Flow Cytometry, Comparison

Journal: Journal of Biological Chemistry

Article Title: Curcumin Suppresses the Induction of Indoleamine 2,3-Dioxygenase by Blocking the Janus-activated Kinase-Protein Kinase Cδ-STAT1 Signaling Pathway in Interferon-γ-stimulated Murine Dendritic Cells

doi: 10.1074/jbc.m807328200

Figure Lengend Snippet: FIGURE 3. JAK1/2 and PKC phosphorylation is important for IDO expres- sion in IFN-stimulated murine BMDCs. A and B, BMDCs were incubated in the presence or absence of either a specific inhibitor of JAK1/2- or PKC (rot- tlerin) for 30 min at the indicated concentration before stimulating with IFN (100 units/ml). Cells were then immediately lysed with TRIzol, and IDO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was detected by RT-PCR. Equal amounts of total cell lysates were also immu- noblotted with anti-IDO Abs. Lower panel, densitometric analysis of each Western blot. Values are the mean S.E. of three experiments. *, p 0.05 compared with LPS-treated BMDCs. C, before incubating with 100 units/ml IFN for 18 h, BMDCs were treated for 30 min with 20 nM Go6976 or 40 M safingol. RT-PCR and immunoblotting were performed as described in A and B, above. D, effects of specific PKC gene silencing on IFN-stimulated BMDCs. The cells were treated with or without a small interfering RNA target- ing the PKC gene for 24 h. Control cells were treated with DOTAP alone. Cells lysates were immunoblotted with anti-PKC, anti-phospho PKC, anti-IDO, and anti--tubulin Abs.

Article Snippet: Polyclonal rabbit anti-mouse Abs against -tubulin, phospho-JAK1, phospho-JAK2, JAK2, phospho-PKC , and PKC were purchased from Santa Cruz Biotechnology, Inc. Generation of BM-derived Murine DCs—DCs were generated frommurine BM cells as described by Inaba et al. (32) with somemodifications.

Techniques: Phospho-proteomics, Incubation, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Control

Journal: Journal of Biological Chemistry

Article Title: Curcumin Suppresses the Induction of Indoleamine 2,3-Dioxygenase by Blocking the Janus-activated Kinase-Protein Kinase Cδ-STAT1 Signaling Pathway in Interferon-γ-stimulated Murine Dendritic Cells

doi: 10.1074/jbc.m807328200

Figure Lengend Snippet: FIGURE 5. Inhibition of JAK1/2 or PKC impairs the binding of phospho- rylated STAT1 to the GAS site of the IRF-1 promoter and inhibits IRF-1 expression in IFN-stimulated murine BMDCs. A, BMDCs were pretreated with either a JAK inhibitor (20 nM) or PKC inhibitor (rottlerin, 5 M) for 1 h and then stimulated with IFN (100 units/ml) for 1 h. A ChIP assay was performed afterimmunoprecipitation(IP)ofchromatinwithanti-STAT1Ab.Immunopre- cipitated chromatin and 1% of input chromatin used for each immunopre- cipitation were subjected to PCR with primers specific for the IRF-1 promoter. B, BMDCs were pretreated with the JAK inhibitor (20 nM), rottlerin (5 M), or both for 1 h and stimulated with IFN for 2 h. Cell lysates were analyzed by Western blotting using Abs against IRF-1 and -tubulin. Relative levels of IRF-1 expression normalized to -tubulin were determined by densitometric scanning of IRF-1 and -tubulin bands. Data shown are representative two independent experiments.

Article Snippet: Polyclonal rabbit anti-mouse Abs against -tubulin, phospho-JAK1, phospho-JAK2, JAK2, phospho-PKC , and PKC were purchased from Santa Cruz Biotechnology, Inc. Generation of BM-derived Murine DCs—DCs were generated frommurine BM cells as described by Inaba et al. (32) with somemodifications.

Techniques: Inhibition, Binding Assay, Expressing, Western Blot

Journal: Leukemia Research and Treatment

Article Title: PKC δ Regulates Translation Initiation through PKR and eIF2 α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

doi: 10.1155/2012/482905

Figure Lengend Snippet: PKC δ regulates the phosphorylation (Ser51) of eIF2 α by ATRA and ATO in APL cells. (a) NB4 cells were left untreated or incubated with ATRA (1 μ M) or ATO (0.4 μ M), for the indicated time periods. Equal amounts of total cell lysate were analyzed by SDS-PAGE and immunoblotted with specific antibodies against PKC δ or p-eIF2 α . β -Actin was used as a loading control. (b) ATRA (1 μ M) induces phosphorylation (Thr505) of PKC δ at the activation domain detected by Western blot in NB4 cells. (c) ATRA (1 μ M) induces PKC δ mRNA expression. After 24 h of ATRA treatment, NB4 cells were collected and total cellular RNA was extracted to detect PKC by RT-PCR using specific primers. The reaction products were analyzed on 2% agarose gels. cDNA synthesis and equal loading were verified by detection of the β -actin transcript. (d) ATRA (1 μ M) induces phosphorylation of eIF2 α in U937 (M4/M5-AML) cells. (e) Inhibition of PKC δ by rottlerin inhibits basal and ATRA-induced phosphorylation (Ser51) of eIF2 α . Exponentially growing NB4 cells were collected and pretreated with specific PKC δ inhibitor rottlerin (4 μ M) for 4 h before adding ATRA (1 μ M) into the culture medium. Equal amount of total cell lysates were analyzed by SDS-PAGE and immunoblotted with specific antibodies against p-eIF2 α , as described in .

Article Snippet: Targeted downregulation of eIF2 α and PKC δ was achieved by using double-stranded small-interfering RNA (siRNA), which were purchased from Santa Cruz Biotechnology and Invitrogen Inc.

Techniques: Phospho-proteomics, Incubation, SDS Page, Control, Activation Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, cDNA Synthesis, Inhibition

Journal: Leukemia Research and Treatment

Article Title: PKC δ Regulates Translation Initiation through PKR and eIF2 α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

doi: 10.1155/2012/482905

Figure Lengend Snippet: PKC δ mediates ATRA-induced phosphorylation (Ser51) of eIF α in normal CD34+HCC and AML cells. (a) All cells were treated with ATRA (1 μ M) in the presence or absence of rottlerin for 24 h and p-(Ser51)-eIF2 α was examined by Western blot analysis. ATRA induced-eIF2 α phosphorylation in human normal CD34+ bone marrow HCC cells. Densitometric analysis shows (right panel) relative expression of p-eIF2 α protein levels after normalization to β -actin expression level. Inhibition of PKC δ by rottlerin blocked the Ser51-phosphorylation eIF2 α in HCC cells (a), HL60 (M2-AML) (b), and THP1 (M4-AML) cells (c).

Article Snippet: Targeted downregulation of eIF2 α and PKC δ was achieved by using double-stranded small-interfering RNA (siRNA), which were purchased from Santa Cruz Biotechnology and Invitrogen Inc.

Techniques: Phospho-proteomics, Western Blot, Expressing, Inhibition

Journal: Leukemia Research and Treatment

Article Title: PKC δ Regulates Translation Initiation through PKR and eIF2 α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

doi: 10.1155/2012/482905

Figure Lengend Snippet: Inhibition of PKC δ blocks ATRA-induced PKR and Ser51-phosphorylation of eIF2 α . (a) Inhibition of PKC δ blocked ATRA-induced phosphorylation (Thr446)/activation of PKR. NB4 cells were pretreated with PKC δ inhibitor rottlerin (4 μ M) for 4 h before adding ATRA (1 μ M). (b) Rottlerin (4 M) inhibits ATRA-induced expression of PKC δ in NB4 cells detected by Western blot analysis. (c) Knockdown of PKC δ by a specific siRNA results in reduction of phosphorylation (Thr446) of PKR in NB4 cells. Bar graph represents inhibition of relative expression of PKC δ (PKC δ / β -actin) and p-PKR (p-PKR/ β -actin) based on densitometric analysis of Western blots.

Article Snippet: Targeted downregulation of eIF2 α and PKC δ was achieved by using double-stranded small-interfering RNA (siRNA), which were purchased from Santa Cruz Biotechnology and Invitrogen Inc.

Techniques: Inhibition, Phospho-proteomics, Activation Assay, Expressing, Western Blot, Knockdown

Journal: Leukemia Research and Treatment

Article Title: PKC δ Regulates Translation Initiation through PKR and eIF2 α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

doi: 10.1155/2012/482905

Figure Lengend Snippet: PKC δ plays a critical role in ATRA-induced terminal differentiation of APL cells. Inhibition of PKC δ blocked ATRA-induced granulocytic differentiation of NB4 cells. NB4 cells were pretreated with rottlerin (4 μ M) for 4 h before adding ATRA (1 μ M). After 48 h of ATRA treatment, cells were collected and analyzed for CD11b positivity as a measure of granulocytic differentiation.

Article Snippet: Targeted downregulation of eIF2 α and PKC δ was achieved by using double-stranded small-interfering RNA (siRNA), which were purchased from Santa Cruz Biotechnology and Invitrogen Inc.

Techniques: Inhibition

Journal: Leukemia Research and Treatment

Article Title: PKC δ Regulates Translation Initiation through PKR and eIF2 α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

doi: 10.1155/2012/482905

Figure Lengend Snippet: mTOR signaling is involved in regulation of (Ser51) of eIF2 α through PKC and PKR. (a) Inhibition of PI3K/Akt/mTOR signaling pathway by rapamycin (20 nM), a specific mTOR inhibitor, led to induction of phosphorylation eIF2 α in NB4 cells. Rapamycin treatment results in induction of p-(Ser51) eIF2 α and PKR and p(Thr446)-PKR in NB4 cells. (b) NB4 cells were pretreated with a specific PI3K inhibitor (LY294002, 20 μ M) induced PKC δ and p-eIF2 α levels. ATRA Equal amounts of total cell lysate were subjected to Western blot analysis to detect p-eIF2 α , p-Akt, and PKC δ expression.

Article Snippet: Targeted downregulation of eIF2 α and PKC δ was achieved by using double-stranded small-interfering RNA (siRNA), which were purchased from Santa Cruz Biotechnology and Invitrogen Inc.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Expressing

Journal: Leukemia Research and Treatment

Article Title: PKC δ Regulates Translation Initiation through PKR and eIF2 α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

doi: 10.1155/2012/482905

Figure Lengend Snippet: Reduced PKC δ and p-eIF2 α protein expression is associated with relapses in AM patients. PKC δ and p-eIF2 α protein expression was assessed by RPPA in 47 paired samples from newly diagnosed and relapsed AML patients to determine if the protein level changes when the disease status changes. The comparison of PKC δ and p-eIF2 α protein expression ((a) and (c)) and distributions ((b) and (d)) of the protein levels between pairs (newly diagnosed and relapsed) were plotted. Data suggest that there is a significant association between the reduced levels of PKC δ ( P = 0.0378) and p-eIF2 α ( P = 0.0041) and relapses in AMP patients.

Article Snippet: Targeted downregulation of eIF2 α and PKC δ was achieved by using double-stranded small-interfering RNA (siRNA), which were purchased from Santa Cruz Biotechnology and Invitrogen Inc.

Techniques: Expressing, Comparison

Journal: Leukemia Research and Treatment

Article Title: PKC δ Regulates Translation Initiation through PKR and eIF2 α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

doi: 10.1155/2012/482905

Figure Lengend Snippet: PKC δ is involved in regulation of translational initiation by eIF2 α through PKR axis in AML cells. ATRA and ATO inhibit translation initiation by inducing inactivation (phosphorylation at Ser-51) of eIF2 α . Suppression of translation initiation is also regulated by other mediators, including induction of translational suppressors (PDCD4 and DAP5) and inhibition of 4E-BP1 [ – ]. The eIF2 α kinases other than PKR, such as GCN2 and PERK, are not involved in regulation of eIF2 α during ATRA-induced differentiation. eIF2 α is involved in regulation of ATRA-regulated cellular proteins in cell cycle arrest (p21 Waf1/Cip1 ), proliferation (c-myc), survival (p70S6K), and apoptosis (DAP5/p97) and differentiation (TG2).

Article Snippet: Targeted downregulation of eIF2 α and PKC δ was achieved by using double-stranded small-interfering RNA (siRNA), which were purchased from Santa Cruz Biotechnology and Invitrogen Inc.

Techniques: Phospho-proteomics, Inhibition

Journal: Oxidative Medicine and Cellular Longevity

Article Title: PKC δ Promotes High Glucose Induced Renal Tubular Oxidative Damage via Regulating Activation and Translocation of p66Shc

doi: 10.1155/2014/746531

Figure Lengend Snippet: PKC δ siRNA inhibits HG-induced p66Shc phosphorylation in HK-2 cells. HK-2 cells were transfected with either PKC δ siRNA or scrambled siRNA and then stimulated with 30 mM HG for 24 hrs. PKC δ , p66Shc, and p-p66Shc were detected by Western blot analysis. An increased expression of p-p66Shc was observed in HK-2 cells induced by HG, but it failed in the group treated with PKC δ siRNA. Values are means ± SEM, N = 4. * P < 0.01 versus LG group, # P < 0.01 versus scrambled siRNA.

Article Snippet: Rottlerin was obtained from Enzo life sciences (USA), mitochondria isolation kit was purchased from Beyotime (Jiangsu, China), 4′,6-diamidino-2-phenylindole 9 (DAPI) was from Sigma-Aldrich (St. Louis, MO), PKC δ siRNA (h) was from Santa Cruz (USA), Lipofectamine 2000 and MitoSOX were from Invitrogen (USA), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) were from Sigma-Aldrich (USA), and DAB kit was from CWBIO (Beijing, China).

Techniques: Phospho-proteomics, Transfection, Western Blot, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: PKC δ Promotes High Glucose Induced Renal Tubular Oxidative Damage via Regulating Activation and Translocation of p66Shc

doi: 10.1155/2014/746531

Figure Lengend Snippet: PKC δ siRNA inhibits p66Shc translocation during HG treatment of HK-2 cells. HK-2 cells were transfected with PKC δ siRNA or scrambled siRNA and treated with HG for different time periods. Lysates of cytosolic and mitochondrial fractions were analyzed for p66Shc and p-p66Shc expression by Western blotting, and β -actin was used as a cytosolic loading control and Cox IV antibody was used as a mitochondrial loading control. Values are means ± SEM, N = 4. * P < 0.01 versus HG group.

Article Snippet: Rottlerin was obtained from Enzo life sciences (USA), mitochondria isolation kit was purchased from Beyotime (Jiangsu, China), 4′,6-diamidino-2-phenylindole 9 (DAPI) was from Sigma-Aldrich (St. Louis, MO), PKC δ siRNA (h) was from Santa Cruz (USA), Lipofectamine 2000 and MitoSOX were from Invitrogen (USA), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) were from Sigma-Aldrich (USA), and DAB kit was from CWBIO (Beijing, China).

Techniques: Translocation Assay, Transfection, Expressing, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: PKC δ Promotes High Glucose Induced Renal Tubular Oxidative Damage via Regulating Activation and Translocation of p66Shc

doi: 10.1155/2014/746531

Figure Lengend Snippet: PKC δ siRNA attenuates the generation of intracellular ROS and mitochondrial superoxide in HK-2 cells exposure to HG. (a) HK-2 cells were stained with dichlorofluorescein-diacetate (DCFH-DA) and MitoSOX, respectively, and then examined by confocal microscopy. Results indicate that PKC δ siRNA exhibits reduced intracellular ROS production in HK-2 cells after treatment with HG (row 1). Similar results were seen in mitochondrial ROS production as assessed by MitoSOX staining (row 2). ((b) and (c)) The bar graphs represent a summary of flow cytometry analyses of cells stained with DCFH-DA and MitoSOX, respectively. Values are means ± SEM, N = 4. * P < 0.01 versus 5 mM D-glucose. # P < 0.01 versus 30 mM D-glucose.

Article Snippet: Rottlerin was obtained from Enzo life sciences (USA), mitochondria isolation kit was purchased from Beyotime (Jiangsu, China), 4′,6-diamidino-2-phenylindole 9 (DAPI) was from Sigma-Aldrich (St. Louis, MO), PKC δ siRNA (h) was from Santa Cruz (USA), Lipofectamine 2000 and MitoSOX were from Invitrogen (USA), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) were from Sigma-Aldrich (USA), and DAB kit was from CWBIO (Beijing, China).

Techniques: Staining, Confocal Microscopy, Flow Cytometry

Journal: Experimental and Therapeutic Medicine

Article Title: Small interfering RNA-mediated knockdown of KRT80 suppresses colorectal cancer proliferation

doi: 10.3892/etm.2020.9306

Figure Lengend Snippet: Knockdown of KRT80 expression decreases the expression levels of cell cycle-related proteins. SW480 cells were transfected with siRNA-KRT80 or siRNA-NC for 48 h. (A) Western blotting was used to analyze the expression levels of KRT80, PPP1CA, p21 and p27. (B) Semi-quantification of the western blotting data in part A. β-actin was used as the internal loading control for the western blotting analysis. Data are presented as the mean ± SD from 3 independent experimental repeats. * P<0.05, ** P<0.01 vs. siRNA-NC group. KRT80, keratin 80; PPP1CA, protein phosphatase 1 catalytic subunit α; p21, cyclin-dependent kinase inhibitor 1A; p27, cyclin-dependent kinase inhibitor 1B; siRNA, small interfering RNA; NC, negative control.

Article Snippet: The separated proteins were subsequently transferred to PVDF membranes (EMD Millipore) and blocked with 5% skimmed milk at room temperature for 1 h. The membranes were incubated with the following primary antibodies overnight at 4˚C: Anti-KRT80 (1:800; cat. no. 16835-1-AP; ProteinTech Group, Inc.), anti-protein phosphatase 1 catalytic subunit α (PPP1CA; 1:2,000; cat. no. 67070-1-Ig; ProteinTech Group, Inc.), anti-cyclin-dependent kinase inhibitor 1A (p21; 1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-cyclin-dependent kinase inhibitor 1B (p27; 1:1,000; cat. no. 3686; Cell Signaling Technology, Inc.) and anti-β-actin (1:2,500; cat. no. A5441; Sigma-Aldrich; Merck KGaA).

Techniques: Expressing, Transfection, Western Blot, Small Interfering RNA, Negative Control

Journal: bioRxiv

Article Title: Characterization of Beta Tubulin Isotypes During Foam Cell Formation

doi: 10.1101/141457

Figure Lengend Snippet: Relative levels of beta tubulin expression were determined by treating cells with PMA (100 nM) and/or LDL (100 ug/ml) for 24 hours, preparing lysates, and performing western blot analysis for each isotype. For each graph, data points resulted from the blotting of 3 different lysates, with each lysate run in duplicate. Bars represent mean +/-SEM. One-way analysis of variance, p< 0.0079 (Beta 1) and p< 0.0208 (Beta 4).

Article Snippet: Human beta tubulin siRNAs were obtained from Santa Cruz Biotechnology: β1 Tubulin siRNA (sc-105000), β3 Tubulin siRNA (sc-105009), and β2C (β4) Tubulin siRNA (sc-105007).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Characterization of Beta Tubulin Isotypes During Foam Cell Formation

doi: 10.1101/141457

Figure Lengend Snippet: THP-1 cells treated with PMA (100nM), representing macrophages, or PMA (100 nM) and LDL (100 ug/ml), representing foam cells, were fixed, permeabilized, incubated in primary mouse anti-beta tubulin antibodies, then Alexa Fluor 647-conjugated goat anti-mouse antibodies, causing the respective beta tubulin isotype to fluoresce. In rows 1 and 3, tubulin appears green, the DAPI-stained nucleus appears blue, and Nile Red-stained lipids appear red. Rows 1 and 3 show all signals merged, while rows 2 and 4 exclusively feature the spatial arrangement of respective beta tubulins.

Article Snippet: Human beta tubulin siRNAs were obtained from Santa Cruz Biotechnology: β1 Tubulin siRNA (sc-105000), β3 Tubulin siRNA (sc-105009), and β2C (β4) Tubulin siRNA (sc-105007).

Techniques: Incubation, Staining

Journal: bioRxiv

Article Title: Characterization of Beta Tubulin Isotypes During Foam Cell Formation

doi: 10.1101/141457

Figure Lengend Snippet: THP-1 cells treated with PMA (100nM), representing macrophages, or PMA (100 nM) and LDL (100 ug/ml), representing foam cells, were fixed, permeabilized, incubated in primary mouse anti-beta tubulin antibodies, then Alexa Fluor 647-conjugated goat anti-mouse antibodies, causing the respective beta tubulin isotype to fluoresce. At least 9 cells per micrograph taken per group for each isotype were then analyzed by measuring the integrated density of the respective beta tubulin fluorescence signal, correcting for background signal, and dividing the corrected fluorescence by the unit area of the cell. On graphs, circles/squares represent mean, and bars represent +/-SEM.

Article Snippet: Human beta tubulin siRNAs were obtained from Santa Cruz Biotechnology: β1 Tubulin siRNA (sc-105000), β3 Tubulin siRNA (sc-105009), and β2C (β4) Tubulin siRNA (sc-105007).

Techniques: Incubation, Fluorescence

Journal: bioRxiv

Article Title: Characterization of Beta Tubulin Isotypes During Foam Cell Formation

doi: 10.1101/141457

Figure Lengend Snippet: Graphs in top row show the beta tubulin:GAPDH ratio in siRNA-treated cells vs. normal cells. Micrographs in row two were cells treated with PMA and LDL but not siRNA. Micrographs in row three were cells transfected with 25 picomoles of siRNA using Santa Cruz Biotechnology transfection reagent for A) β1 tubulin, B) β3 tubulin, and C) β4 tubulin, left to incubate for 5 hours, then treated for 24 hours with PMA (100 nM) and LDL (100 ug/ml). Twenty-four hours after the addition of PMA and LDL, cells were stained with Oil Red O; red spots within cells are stained lipids. All cell microscopy was taken at a magnification of 400X.

Article Snippet: Human beta tubulin siRNAs were obtained from Santa Cruz Biotechnology: β1 Tubulin siRNA (sc-105000), β3 Tubulin siRNA (sc-105009), and β2C (β4) Tubulin siRNA (sc-105007).

Techniques: Transfection, Staining, Microscopy

Journal: bioRxiv

Article Title: Characterization of Beta Tubulin Isotypes During Foam Cell Formation

doi: 10.1101/141457

Figure Lengend Snippet: THP-1 cells were transfected with 25 picomoles of siRNA using Santa Cruz Biotechnology transfection reagent and treated for 24 hours with either PMA (100 nM) or PMA and LDL (100 ug/ml). After 24 hours, cells were stained with Oil-Red O to facilitate microscopy. All microscopy pictures were taken at a magnification of 400X. At least 9 cells in each micrograph taken for each treatment group were then outlined in ImageJ, and evaluated for area. In graphs, shapes represent mean, and bars represent +/-SEM.

Article Snippet: Human beta tubulin siRNAs were obtained from Santa Cruz Biotechnology: β1 Tubulin siRNA (sc-105000), β3 Tubulin siRNA (sc-105009), and β2C (β4) Tubulin siRNA (sc-105007).

Techniques: Transfection, Staining, Microscopy

Journal: bioRxiv

Article Title: Characterization of Beta Tubulin Isotypes During Foam Cell Formation

doi: 10.1101/141457

Figure Lengend Snippet: THP-1 Cells were treated with 24 hours with PMA (100 nM) and LDL (100 g/ml) following their respective transfections. The cells in row 2 were transfected with 25 picomoles of SiRNA using Santa Cruz Biotechnology transfection reagent for A) β1 Tubulin B) β3 Tubulin, and C) β4 Tubulin for five hours prior to incubation in PMA and LDL. Cells in row 3 were transfected with 500 ng of overexpression plasmid containing the full-length cDNA for A) β1 Tubulin B) β3 Tubulin, and C) β4 Tubulin for five hours before treatment with PMA and LDL. 24 hours after the addition of treatment, cells were stained with Oil Red O; red spots within cells are stained lipids. All cell microscopy was taken at a magnification of 400x.

Article Snippet: Human beta tubulin siRNAs were obtained from Santa Cruz Biotechnology: β1 Tubulin siRNA (sc-105000), β3 Tubulin siRNA (sc-105009), and β2C (β4) Tubulin siRNA (sc-105007).

Techniques: Transfection, Incubation, Over Expression, Plasmid Preparation, Staining, Microscopy

Journal: bioRxiv

Article Title: Characterization of Beta Tubulin Isotypes During Foam Cell Formation

doi: 10.1101/141457

Figure Lengend Snippet: THP-1 cells were transfected with 500 ng of overexpression plasmid containing the full-length cDNA for A) β1 Tubulin B) β3 Tubulin, and C) β4 Tubulin for before treatment with either PMA (100 nM) or PMA and LDL (100 ug/mL) for 24 hours. After 24 hours, cells were stained with Oil Red O to facilitate microscopy. All microscopy pictures were taken at a magnification of 400X. At least 9 cells in each micrograph per treatment group were then outlined in ImageJ, and evaluated for surface Area. In graphs, shapes represent mean, and bars represent +/-SEM.

Article Snippet: Human beta tubulin siRNAs were obtained from Santa Cruz Biotechnology: β1 Tubulin siRNA (sc-105000), β3 Tubulin siRNA (sc-105009), and β2C (β4) Tubulin siRNA (sc-105007).

Techniques: Transfection, Over Expression, Plasmid Preparation, Staining, Microscopy

Journal: FEBS Open Bio

Article Title: Respiratory syncytial virus (RSV) suppression of glucocorticoid receptor phosphorylation does not account for repression of transactivation ☆

doi: 10.1016/j.fob.2013.07.005

Figure Lengend Snippet: Knock-down of protein phosphatases does not reverse RSV repression of dexamethasone-induced GILZ. A549 cells were transfected with siRNA against PP5, PP1 and PP2A or a control siRNA. 48 h later they were RSV (MOI = 3)- (black) or mock-infected (white) and treated with vehicle or 100 nM dexamethasone for 5 h. Dexamethasone-induced GILZ mRNA was determined by real-time PCR and normalized to GAPDH and CAP-1 (A). % repression by RSV was calculated as previously (B). siRNA knockdown was confirmed by real-time PCR (C–E). Means and SD are shown ( n = 4).

Article Snippet: 50 nM protein phosphatase 5 (PP5), pan protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A-Cα) siRNA and control siRNA-A (Santa Cruz Biotechnology; sc-44602, sc-43545, sc-43509 and sc-37007, respectively) was transfected into A549 cells using DharmaFECT 2 (Thermo Fisher Scientific Inc.,) according to the manufacturer's instructions.

Techniques: Knockdown, Transfection, Control, Infection, Real-time Polymerase Chain Reaction