Journal: Oncotarget

Article Title: NOD2 up-regulates TLR2-mediated IL-23p19 expression via NF-κB subunit c-Rel in Paneth cell-like cells

doi: 10.18632/oncotarget.11467

Figure Lengend Snippet: PC-like cells were pretreated for 30 min with small molecule inhibitors BAY117082 (10 μM; NF-κB), SB203580 (20 μM; p38 MAPK), SP600125 (50 μM; JNK), and U0126 (50 μM; ERK1/2), LY294002 (50 μM; PI3K) and subsequently were stimulated for 4 h with PGN (10 μg/ml). Total RNA was isolated and IL-23p19 mRNA expression was determined by real-time PCR. B. Effect of PGN on activation of IκBα in the NF-κB pathway in PC-like cells. PC-like cells were stimulated with PGN (10 μg/ml), and then whole-cell extracts were prepared at the indicated time points and were analyzed for phospho-IκBα and IκBα by western blot. Top, quantitative analysis of proteins; bottom, representative immunoblot images. Data are shown as means ± SD of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Small molecule inhibitors (BAY117082, LY294002, SB203580, U0126 and SP600125) were purchased from InvivoGen.

Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: Targeted inhibition of CDK7 reverses chemoresistance in pancreatic cancer. A Western blotting of TB32047 and MIA PaCa-2 gemcitabine- and paclitaxel-resistant cells (TBGPR, MIAR) and parental cells (TBWT, MIAWT). B The CDK7 inhibitor THZ1 synergistically enhances the antiproliferative effects of GEM and PTX in TBGPR and MIAGPR cells. The proliferation was determined after 72 h of treatment with THZ1 and GEM/PTX alone or in combination. Below is a plot of the fraction impacted versus the combination index. CI < 1.0, synergism. C Diagrammatic representation of the research outcome. Gemcitabine and paclitaxel combined with CDK7 inhibition induces DNA damage, G2-M arrest, and subsequent apoptosis

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: CRISPR-Cas9 knockout screens to identify chemosensitivity targets in pancreatic cancer cells treated with gemcitabine and paclitaxel. A Schematic diagram of the timeline and experimental procedure for CRISPR-Cas9 screening using protein kinase library. B, C Volcano plots showing log 2 -transformed sgRNA normalized fold change and -log 10 -transformed p -value of gemcitabine ( B ) and paclitaxel ( C ) versus vehicle-treated TB32047 cells, with significant genes in positive and negative selection highlighted in red and blue, respectively. D Venn diagram showing the shared genes of the top 20 candidate genes for gemcitabine and paclitaxel negative screening, which are Cdk7, Wee1, Fgfrl1, and Havcr2. E Scatterplots showing log 2 -transformed sgRNA normalized read counts of gemcitabine (left) and paclitaxel (right) versus vehicle-treated TB32047 cells, with sgRNAs targeting Cdk7 highlighted. F Data from TCGA-PDAC were applied for survival analysis. Kaplan–Meier survival analysis shows that higher CDK7 mRNA expression is associated with a tendency toward poor overall survival. G The analysis of CDK7 expression in PDAC tissues and normal pancreatic tissues was conducted using the TCGA (PDAC) and GTEx (Pancreas) databases. Taken together, the results indicate that CDK7 expression levels were significantly higher in pancreatic cancer than in the corresponding normal pancreatic tissue. H Western blot analysis revealed a variation in CDK7 expression between pancreatic cancer subtypes. * p < 0.05; *** p < 0.001, and **** p < 0.0001 were calculated using the unpaired Student’s t test

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: CRISPR, Knock-Out, Transformation Assay, Selection, Expressing, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: CDK7 knockout in combination with gemcitabine and paclitaxel treatment inhibits proliferation and increases apoptosis in pancreatic cancer cells. A Western blotting of TB32047 and MIA PaCa-2 cells transfected with CDK7 sgRNAs or non-targeting control. B The dose–response curves for GEM and PTX in TB32047 and MIA PaCa-2 CDK7 KO or NC cells. Seventy-two hours following treatment, cell survival was measured and normalized. Data are presented as the mean of three independent experiments ( n = 3). C–F CDK7-WT (NC sgRNA) and CDK7-knockout TB32047 ( C , D ) cells, as well as MIA PaCa-2 ( E , F ) cells, were treated with vehicle or chemotherapy for 24 h and then analyzed for apoptosis using flow cytometry. Data are presented as the mean of three independent experiments ( n = 3). ** p < 0.01; *** p < 0.001, and **** p < 0.0001 by two-way ANOVA with a Tukey multiple comparison test. G and H TB32047 ( G ) and MIA PaCa-2 ( H ) cells expressing CDK7 or control sgRNAs were treated with GEM and PTX; caspase 3/7 activity was measured 24 h later. Data are presented as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by two-way ANOVA with a Tukey multiple comparison test. I . Western blot analysis of TB32047 (left) and MIA PaCa-2 (right) cells transfected with non-targeting control (NC sgRNA) or CDK7-targeting sgRNAs (CDK7-sg1, CDK7-sg2, CDK7-sg3, CDK7-sg4) and treated for 24 h with vehicle or GEM plus PTX

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Knock-Out, Western Blot, Transfection, Control, Flow Cytometry, Comparison, Expressing, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: Targeted inhibition of CDK7 enhanced gemcitabine and paclitaxel chemotherapy response in pancreatic cancer in vitro. A Dose–response curves of TB32047 and MIA PaCa-2 wild-type cells to THZ1. Cell proliferation was evaluated 72 h after drug treatment. Data are presented as the mean of three independent experiments (n = 3). B and C The CDK7 inhibitor THZ1 enhances the antiproliferative impact of GEM and PTX in pancreatic cancer cells synergistically. Proliferation analysis was performed on TB32047 ( B ) and MIA PaCa-2 ( C ) cells treated for 72 h with THZ1 and GEM/PTX alone or in combination. The plot of fraction affected versus combination index is shown below the x-axis. CI < 1.0, synergism. D and F Clonogenic assay of TB32047 ( D ) and MIA PaCa-2 ( F ) cells treated with vehicle or drugs for 72 h and cultured without drugs for an additional 7–10 days. The cells remaining after the treatment were fixed and stained with crystal violet. Representative images (left) and quantifications (right) of three independent experiments ( n = 3) are shown. * p < 0.05 and ** p < 0.01 by one-way ANOVA with a Tukey multiple comparison test. ns: not significant. E and G . Caspase 3/7 activity was evaluated in TB32047 ( E ) and MIA PaCa-2 ( G ) cells treated for 72 h with vehicle or drugs. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA with a Tukey multiple comparison test. H and I Flow cytometry-based apoptotic assay of TB32047 and MIA PaCa-2 cells treated for 72 h with drugs or drug combination for 72 h. Representative images ( H ) and quantification ( I ) of three independent experiments ( n = 3) are shown. ** p < 0.005 by two-way ANOVA with a Tukey multiple comparison test

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, In Vitro, Clonogenic Assay, Cell Culture, Staining, Comparison, Activity Assay, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: Combined CDK7 inhibition with standard chemotherapy suppresses PDAC tumor growth in vivo. A Images of TB32047 orthotopic mouse model of pancreatic cancer after treatment for 16 days. B and C Relative tumor weights ( B ) and volumes ( C ) of TB32047 orthotopic pancreatic cancer mouse model after treatment. *** p < 0.001 and **** p < 0.0001 by one-way ANOVA with a Tukey multiple comparison test. ns: not significant. D Western blot analysis of tissue samples from mice with pancreatic cancer that had been treated with vehicle, GEM/nab-PTX, or a combination. E Hematoxylin and eosin (H&E) staining demonstrated that the combination of THZ1 and GEM with nab-PTX decreased tumor cell content compared to the group receiving single components and the control group. Scale bar (black), 50 μm

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, In Vivo, Comparison, Western Blot, Staining, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: CDK7 inhibition induces cell cycle arrest, apoptotic cell death and DNA damage through the STAT3-MCL1-CHK1 axis. A Gene set enrichment analysis (GSEA) on the RNA-sequencing data of BXPC-3, Mia PaCa-2, and PANC-1 cells treated with THZ1, a selective CDK7 inhibitor. Transcriptomic gene expression data are based on the GEO dataset GSE121273. B Western blotting of TB32047 and MIA PaCa-2 cells treated with vehicle (DMSO), THZ1, and GEM plus PTX, alone or in combination, for 24 h. C Immunofluorescence of TB32047 cells treated with vehicle (DMSO), THZ1, and GEM/PTX, alone or in combination, for 24 h. Cells were subsequently stained with antibodies to γH2AX (green) and DAPI (blue; nuclei). Scale bar (white), 20 μm

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, RNA Sequencing Assay, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Biochemical pharmacology

Article Title: Expression and genotype-dependent catalytic activity of N -acetyltransferase 2 (NAT2) in human peripheral blood mononuclear cells and its modulation by Sirtuin 1

doi: 10.1016/j.bcp.2018.08.034

Figure Lengend Snippet: NAT2 expression on PBMC. a. NAT2 total protein expression is plotted as percentage of NAT2-positive cells for all PBMC (filled circle), T cells (filled square), B cells (filled triangle), NK cells (inverted filled triangle). The data presented are the mean ± SEM of twenty independent experiments, ***p < 0.001 (One-way ANOVA, Tukey-Kramer multiple comparison post-hoc test). b. NAT2 protein expression according to NAT2 predicted phenotype; rapid acetylators (filled circle), intermediate acetylators (filled square), and slow acetylators (filled triangle). The data presented are the mean ± SEM of twenty independent experiments, *p<0.05, **p<0.01, ***p < 0.001 (One-way ANOVA, Tukey-Kramer multiple comparison post-hoc test) c. SIRT1 total protein expression is plotted as percentage of SIRT1-positive cells for all PBMC (filled circle), T cells (filled square), B cells (filled triangle), NK cells (inverted filled triangle). The data presented are the mean ± SEM of twenty independent experiments, ***p < 0.001 (One-way ANOVA, Tukey-Kramer multiple comparison post-hoc test). d. SIRT1 protein expression according to NAT2 predicted phenotype; rapid acetylators (filled circle), intermediate acetylators (filled square), and slow acetylators (filled triangle). The data presented are the mean ± SEM of twenty independent experiments (One-way ANOVA, Tukey-Kramer multiple comparison post-hoc test).

Article Snippet: Cells were treated with increasing concentrations of the small molecule SIRT1 activity enhancer SRT1720 (0.1 – 1 μM) (Santa Cruz Biotechnology, Dallas, TX, USA), and incubated for 3 h at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Expressing

Journal: Biochemical pharmacology

Article Title: Expression and genotype-dependent catalytic activity of N -acetyltransferase 2 (NAT2) in human peripheral blood mononuclear cells and its modulation by Sirtuin 1

doi: 10.1016/j.bcp.2018.08.034

Figure Lengend Snippet: Effect of SIRT1 activity enhancing on INH N-acetylation activity. a. PBMC were treated in presence of SRT1720 and cultured in presence of INH as described in materials and methods. Quantification of acetyl-INH formation on PBMC culture media supernatants were detected by HPLC. The data presented are the mean ± SEM of twenty independent experiments, ***p < 0.001 (One-way ANOVA, Tukey-Kramer multiple comparison post-hoc test). b. Comparison between untreated cells (filled circle), cells treated with 0.1 μM SRT1720 (filled square), 0.5 μM SRT1720 (filled triangle), and 1.0 μM SRT1720 (inverted filled triangle). Cells treated with 0.5 μM SRT1720 showed an apparent Vmax of 20.4 ±1.9 nM Ac-INH/24h/million cells; cells treated with 1.0 μm of SRT1720, decreased as well as the Vmax for INH N-acetylation was reduced to 12.7 ±0.8 nM Ac-INH/24h/million cells. The data presented are the mean ± SEM of twenty independent experiments, ****p < 0.0001 (Non-linear regression). c. INH N-acetylation in rapid (filled square), intermediate (filled triangle) and slow (inverted filled triangle) acetylators subjects following treatment with 1.0 μM SRT1720. Rapid acetylators showed an apparent Vmax of 30.0 ±1.3 nM Ac-INH/24h/million cells; intermediate acetylators showed an apparent Vmax of 9.93 ±1.22 nM Ac-INH/24h/million cells; slow acetylators showed an apparent Vmax of 8.55 ±0.83 nM Ac-INH/24h/million cells. The apparent Vmax in NAT2 intermediate, and slow acetylators was significantly lower compared to rapid acetylators (p<0.0001). The data presented are the mean ± SEM of twenty independent experiments, ****p < 0.0001 (Non-linear regression).

Article Snippet: Cells were treated with increasing concentrations of the small molecule SIRT1 activity enhancer SRT1720 (0.1 – 1 μM) (Santa Cruz Biotechnology, Dallas, TX, USA), and incubated for 3 h at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Cell Culture

Journal: Biochemical pharmacology

Article Title: Expression and genotype-dependent catalytic activity of N -acetyltransferase 2 (NAT2) in human peripheral blood mononuclear cells and its modulation by Sirtuin 1

doi: 10.1016/j.bcp.2018.08.034

Figure Lengend Snippet: Effect of SIRT1 activity silencing on INH N-acetylation. a. SIRT1 expression silencing on PBMC using siRNA. Protein expression was significantly lower after 24h treatment. The data presented are the mean ± SEM of three independent experiments, ***p < 0.001 (One-way ANOVA, Tukey-Kramer multiple comparison post-hoc test). b. PBMC were treated with SIRT1 siRNA and cultured in presence of INH as described in materials and methods. Quantification of acetyl-INH formation on PBMC culture media supernatants were detected by HPLC. Increased INH N-acetylation was observed for the treated cells vs the untreated. The data presented are the mean ± SEM of three independent experiments, **p < 0.01 (One-way ANOVA, Tukey-Kramer multiple comparison post-hoc test).

Article Snippet: Cells were treated with increasing concentrations of the small molecule SIRT1 activity enhancer SRT1720 (0.1 – 1 μM) (Santa Cruz Biotechnology, Dallas, TX, USA), and incubated for 3 h at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Expressing, Cell Culture

Journal: Nature

Article Title: Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy

doi: 10.1038/nature25013

Figure Lengend Snippet: (a) Retinal digest preparations; acellular capillaries are marked by arrows. (b–e) Quantitative retinal morphometry of images in a; i.e., endothelial cells (EC, b), pericytes (PC, c), extravascular pericytes (Ev-PC, d), and acellular capillaries (ac-Cap, e). (f) Fluorescent images of FITC-BSA 1 hour after injection. (g) Quantification of leaked FITC-BSA. (h) N- and VE-cadherin expression in retinas from wild-type and Ins2Akita littermates treated with or without sEH inhibitor (sEH-I). For gel source data, see Supplementary Fig. 1. (i) N-cadherin (green) and desmin (red) in the primary vascular layer and second capillary layer. (j) VE-cadherin in the retinal vasculature. Scale bar 20μm. n = 5 (f–h) or 6 (a–e, i, j) mice per group. Data are mean ± s.e.m. P values determined by 2way ANOVA.

Article Snippet: In some experiments pericytes were treated with small interfering RNA directed against N-cadherin (sc-29403, Santa Cruz) or an N-cadherin overexpression plasmid (addgene #18870).

Techniques: Injection, Expressing

Journal: Nature

Article Title: Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy

doi: 10.1038/nature25013

Figure Lengend Snippet: (a) VE-cadherin in endothelial cells treated with solvent, 19,20-EDP/DHDP or VEGF. Arrowheads indicate the discontinuous VE-cadherin pattern. (b) Permeability of human endothelial cells to dextran with different molecular masses. (c) Transendothelial electrical resistance (TEER) in murine brain endothelial cells. (d–e) Internalized VE-cadherin (yellow) in endothelial cells. (f) Surface and internalised VE-cadherin in endothelial cells. (g) N- and VE-cadherin expression in endothelial-pericyte co-cultures. (h) Pericyte mobility on endothelial cells after N-cadherin downregulation (N-si), N-cadherin overexpression (N-OV), or treatment with EDP/DHDP; n = 120 cells per group. (i–j) Internalized N-cadherin (yellow) in human pericytes. (k) Migrating pericytes (arrowheads) in ex vivo retinal whole mounts. (l) Association of PS1 (PS1-F: full length; PS1-N: N-terminal fragment) with VE- and N-cadherin immunoprecipitated from endothelial cell-pericyte co-cultures. (m) VE-cadherin (green), PS1 (red) and N-cadherin (blue) in retinas from 12 month old mice treated with vehicle or sEH inhibitor (sEH-I). Arrows indicate disrupted VE-cadherin pattern. (n) Co-precipitation of PS1 with cholesterol from endothelial cell-pericyte co-cultures. (o) Co-precipitation of VE- and N-cadherins with cholesterol from endothelial cell-pericyte co-cultures. (p) CH2 resonance centerband intensities of 1H MAS NMR temperature scans of isolated brain plasma membranes from wild-type and sEH−/− mice exposed to 19,20-EDP (100 μmol/L) or 19,20-DHDP (10 or 100 μmol/L). For gel source data, see Supplementary Fig. 1. Scale bars 20μm. n = 4 (a, f–h, l, n–o), 5 (i–j), 6 (b, d–e), 8 (c) independent experiments, or 6 retinas (k, m) per group. Data are mean ± s.e.m. P values determined by 2way ANOVA (b) or 1way ANOVA (c, e–f, h, j–k).

Article Snippet: In some experiments pericytes were treated with small interfering RNA directed against N-cadherin (sc-29403, Santa Cruz) or an N-cadherin overexpression plasmid (addgene #18870).

Techniques: Permeability, Expressing, Over Expression, Ex Vivo, Immunoprecipitation, Isolation

Journal: Nature

Article Title: Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy

doi: 10.1038/nature25013

Figure Lengend Snippet: (a) sEH (red) in wild-type mice retinas 7 days after intravitreal injection of GFP track adenoviral-sEH (sEHWT). (b) Retinal sEH activity 14 days after intravitreal injection of adenoviruses encoding GFP, sEHWT or the sEHΔEH mutant. (c) 19,20-EDP/DHDP levels in retinas 14 days after adenoviral injection to animals receiving vehicle or the sEH inhibitor (sEH-I). (d) Retinal digest preparations 14 days after adenovirus injection with or without sEH inhibitor (+I) treatment. Acellular capillaries are marked by arrowheads. (e) Vascular permeability (titrc-dextrin; red) 14 days after virus administration. (f–i) Quantitative retinal morphometry of images in d; i.e., endothelial cells (EC, f), pericytes (PC, g), extravascular pericytes (Ev-PC, h), and acellular capillaries (ac-Cap, i); n = 6 (GFP), 9 (sEHWT) or 8 (sEHWT+I, sEHΔEH) mice per group. (j) Quantification of leaked Titrc-dextrin. (k) N-cadherin (cyan) and desmin (red) in retinas treated with adenoviruses. Note the perivascular desmin positive protrusions that mark extravascular pericytes in sEHWT treated retinas. (l) VE-cadherin (green) and collagen IV (red) in retinas 14 days after adenovirus injection. Capillaries with abnormal VE-cadherin indicated with arrowheads. Scale bars 50μm. n = 5 (b), 6 (e, j–l) mice or 6 biologically independent samples per group (c). Data are mean ± s.e.m. P values determined by 1way ANOVA.

Article Snippet: In some experiments pericytes were treated with small interfering RNA directed against N-cadherin (sc-29403, Santa Cruz) or an N-cadherin overexpression plasmid (addgene #18870).

Techniques: Injection, Activity Assay, Mutagenesis, Permeability

Journal: International Journal of Oncology

Article Title: Oncogenic role of abnormal spindle-like microcephaly-associated protein in lung adenocarcinoma

doi: 10.3892/ijo.2021.5203

Figure Lengend Snippet: ASPM promotes lung adenocarcinoma cell proliferation, migration and invasion in vitro . (A) Reverse transcription-quantitative PCR was used to confirm ASPM-knockdown using two siRNAs in H1299 cells. (B) Cell Counting Kit-8 and (C) EDU assays were performed to identify proliferation after ASPM-knockdown in H1299 cells (scale bar, 100 µ m). (D) Wound healing (scale bar, 100 µ m), migration and invasion (scale bar, 50 µ m) assays were performed to identify the migratory and invasive abilities after ASPM-knockdown in H1299 cells. (E) Changes in the expression levels of the epithelial-mesenchymal transition biomarkers E-cadherin, N-cadherin and Snail after ASPM-knockdown were detected by western blotting. Data are presented as the mean ± SD (n=3). * P<0.05, ** P<0.01 and *** P<0.001 vs. scramble. ASPM, abnormal spindle-like microcephaly; si-RNA, small interfering RNA; OD, optical density; EDU, 5-Ethynyl-2′-deoxyuridine.

Article Snippet: After blocking with 5% milk in TBS-Tween (TBST; 0.1% Tween 20) for 1 h at room temperature, membranes were incubated with primary antibodies against ASPM (1:500; cat. no. sc-488883; Santa Cruz Biotechnology, Inc.), GAPDH (used as a reference protein; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 5174), E-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 14472), N-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 13116), Snail (1:1,000; Cell Signaling Technology, Inc.; cat. no. 3879), PI3K p110α (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4249), phosphorylated (p) AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4060) and AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4691) overnight at 4°C.

Techniques: Migration, In Vitro, Reverse Transcription, Real-time Polymerase Chain Reaction, Knockdown, Cell Counting, Expressing, Western Blot, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Oncogenic role of abnormal spindle-like microcephaly-associated protein in lung adenocarcinoma

doi: 10.3892/ijo.2021.5203

Figure Lengend Snippet: ASPM promotes lung adenocarcinoma cell proliferation and metastasis in vivo . (A) Representative images of tumors from different treatment groups 5 weeks after tumor cell injections. (B) Tumor volume and (C) weight were lower for xenograft tumors with ASPM-knockdown than for xenograft tumors with shNC. (D) Representative images of immunohistochemical staining for Ki67, E-cadherin and N-cadherin in xenograft tumors (magnification, ×100). (E) Representative images of lung metastasis. (F) Bar chart of lung metastasis nodules in shNC and shASPM groups. (G) Representative images of HE staining of lung metastasis in shNC and shASPM groups (magnification, ×100 and ×200). * P<0.05; ** P<0.01. ASPM, abnormal spindle-like microcephaly; sh, short hairpin RNA; NC, negative control; HE, hematoxylin and eosin.

Article Snippet: After blocking with 5% milk in TBS-Tween (TBST; 0.1% Tween 20) for 1 h at room temperature, membranes were incubated with primary antibodies against ASPM (1:500; cat. no. sc-488883; Santa Cruz Biotechnology, Inc.), GAPDH (used as a reference protein; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 5174), E-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 14472), N-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 13116), Snail (1:1,000; Cell Signaling Technology, Inc.; cat. no. 3879), PI3K p110α (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4249), phosphorylated (p) AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4060) and AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4691) overnight at 4°C.

Techniques: In Vivo, Knockdown, Immunohistochemical staining, Staining, shRNA, Negative Control

Journal: International Journal of Oncology

Article Title: Oncogenic role of abnormal spindle-like microcephaly-associated protein in lung adenocarcinoma

doi: 10.3892/ijo.2021.5203

Figure Lengend Snippet: Activation of the PI3K/AKT signaling pathway restores the migratory and invasive abilities of shASPM-transfected lung adenocarcinoma cells. (A) Changes in epithelial-mesenchymal transition biomarker expression, including N-cadherin, E-cadherin and Snail, were detected in ASPM-knockdown H1299 and A549 cells with or without 740 Y-P treatment for 24 h. Wound healing (scale bar, 100 µ m), migration and invasion assays (scale bar, 50 µ m) in ASPM-knockdown (B) H1299 and (C) A549 cells with or without 740 Y-P treatment for 24 h. Data are presented as the mean ± SD (n=3). * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. ASPM, abnormal spindle-like microcephaly; sh, short hairpin RNA; NC, negative control; p, phosphorylated.

Article Snippet: After blocking with 5% milk in TBS-Tween (TBST; 0.1% Tween 20) for 1 h at room temperature, membranes were incubated with primary antibodies against ASPM (1:500; cat. no. sc-488883; Santa Cruz Biotechnology, Inc.), GAPDH (used as a reference protein; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 5174), E-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 14472), N-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 13116), Snail (1:1,000; Cell Signaling Technology, Inc.; cat. no. 3879), PI3K p110α (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4249), phosphorylated (p) AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4060) and AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 4691) overnight at 4°C.

Techniques: Activation Assay, Transfection, Biomarker Discovery, Expressing, Knockdown, Migration, shRNA, Negative Control

Journal: Molecular Cancer

Article Title: Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing

doi: 10.1186/s12943-023-01799-7

Figure Lengend Snippet: Comparison of Homology-Directed Recombination versus Homology-independent-targeted-insertion for targeted knock-in of a GD2-CAR into TRAC . a Schematic overview of workflow for experiments in b-d, and nanoplasmid designs for knock-in templates. b - d head-to-head comparison of constructs HDR2c, HITI2c and HITI1c. 5 × 10 6 cells were electroporated per condition on day 2 post activation using respective constructs (0.75 µg of nanoplasmid per 1 × 10. 6 cells) and analyzed via flow cytometry on day 10 ( b , representative donor; c , pooled frequencies) and counted on the same day to assess GD2-CAR-T cell counts ( d ) ( n = 4 independent donors). e – h HDR inhibitor induced modulation of GD2-CAR-T cell integration via HITI and HDR. e Schematic related to f–h. f Representative histograms of GD2-CAR expression after CRISPR knock-in with HDR2c or HITI1c templates either left untreated or treated with 1 µM of AZD0156 for 18 h post electroporation. g + h GD2-CAR expression ( g ) and GD2-CAR-T cell counts ( h ) normalized to untreated CRISPR knock-in samples after 18 h of treatment with indicated concentrations of AZD0156 ( n = 3 independent donors). i-j, CRISPR knock-in of non-activated T cells using HITI1c and HDR2c for knock-in of the GD2-CAR. Indicated are knock-in frequencies ( i ), Viability ( j ) and GD2-CAR-T cell yield. Cells were counted and analyzed via flow cytometry on day 10 or 14 ( n = 4 independent donors). p values were determined by paired two-tailed t tests. Error bars indicate standard deviation (SD)

Article Snippet: Small molecule inhibitors AZD0156 (Selleck Chemicals) and Methotrexate (Sigma Aldrich) were resuspended in DMSO and added to the cell culture media as indicated.

Techniques: Comparison, Knock-In, Construct, Activation Assay, Flow Cytometry, Expressing, CRISPR, Electroporation, Two Tailed Test, Standard Deviation