Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 2. KRASG12D increases SLC25A1 expression by activating GLI1. A, Consensus GLI1 binding site in SLC25A1 promoter. The reported consensus GLI1 binding site is GACCACCCA. The putative GLI1 binding site was identified in SLC25A1 promoter. P1 primers (red) are designed to flank the putative GLI1 binding sequence, while P2 primers (green) are designed for detecting negative binding as a control. The length of the detected fragment, including the binding site, is 0.2 kb. B, Correlative expression of GLI1 and SLC25A1 in 179 pancreatic adenocarcinoma tissues based on GEPIA analysis of PAAD dataset. The correlation coefficient was calculated by Spearman rank for R ¼ 0.66. C, PANC1 cells were used for a ChIP assay after GLI1 immunoprecipitation, with primers that flanked the putative GLI1 binding sequence (P1) and non-binding sites (P2). The enrichment was assayed by qRT-PCR. Also, the RT-PCR products at the end of 40 cycles were analyzed by agarose gel electrophoresis (bottom). D, IHC staining of GLI1 on pancreatic tissue sections of fElasCreERT and KrasG12D/þ mice (n ¼ 5; 400). E, Quantitation of GLI1 level in D. F, Western blot analysis of GLI1 in the pancreata of fElasCreERT and KrasG12D/þ mice (n ¼ 3). G, qRT-PCR of GLI1 in the pancreata of fElasCreERT and KrasG12D/þ mice (n ¼ 5). H, Western blot analysis of GLI1 in HPDE cells and pancreatic cancer BxPC3, MIA PaCa-2, AsPC1, PANC1, SW1990, and Su.86.86 cells using the stripped membrane from Fig. 1E. b-Actin was used as loading control as in Fig. 1E. I, Western blot analysis of GLI1 in cytosol and nucleus of HPDE, ASPC1, and PANC1 cells. J, PANC1 cells with or without GANT61 treatment at indicated concentrations for 72 hours and then the cells were harvested to isolate mRNA. The mRNA of SLC25A1 was analyzed by qRT-PCR. K, PANC1 cells were treated with different concentrations of GANT61 for 72 hours and the cell lysates were subjected to Western blot analysis for SLC25A1, GLI1, FASN, and ACSL1. L, PANC1 cells were treated with 20 mmol/L of GANT61 for 4 hours and the cells were collected for GLI1 ChIP assay with primers that flanked the putative GLI binding sequence. The results were analyzed by qPCR. PCR products were analyzed by agarose gel electrophoresis (bottom). M, Western blot analysis of the levels of GLI1 and SLC25A1 in wild-type (WT) and GLI1 knockout AsPC1 cells. N, Representative image (left) and quantification (right) of Transwell migration assays in wild-type and GLI1 knockout AsPC1 cells. O, Representative image (left) and quantification (right) of Transwell migration assaysin AsPC1 cells treated with or without GANT61 (30 mmol/L).Results are expressed as mean SD and were statistically evaluatedwith a t test. ns, not significant; , P < 0.05; , P < 0.01; , P < 0.0001.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Sequencing, Control, Immunoprecipitation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Immunohistochemistry, Quantitation Assay, Western Blot, Membrane, Knock-Out, Migration

Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 3. HFD challenge stimulates the KRAS-GLI1 axis to induce SLC25A1 expression. A, H&E staining and IHC staining of vimentin in pancreatic tissue sections of KrasG12D/þ mice fed with ND or high HFD (n ¼ 5; 200x). B, Quantitation of vimentin level in A. C, IHC staining of GLI1 (400) and SLC25A1 (200) on pancreatic tissue sections of KrasG12D/þ mice fed with ND or HFD (n ¼ 5). D, Quantitation of GLI1 and SLC25A1 levels in C. E, Western blot analysis for SLC25A1 and GLI1 in the pancreata of KrasG12D/þ mice fed with ND or HFD (n ¼ 3). F, qRT-PCR analysis of Slc25a1 gene expression in the pancreata of KrasG12D/þ mice fed with ND or HFD (n ¼ 5). G, IHC staining of ACSL1 (200) and FASN (200) on pancreatic tissue sections of KrasG12D/þ mice fed with ND or HFD (n ¼ 5). H, Quantitation of ACSL1 and FASN levels in G. I, Western blot analysis of FASN and ACSL1 in the pancreata of fElasCreERT and KrasG12D/þ mice fed with ND or HFD. J and K, Total citrate and FA levels in pancreatic tissues from KrasG12D/þ mice fed with ND or HFD. Results are expressed as mean SD and were statistically evaluated with a t test. , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Expressing, Staining, Immunohistochemistry, Quantitation Assay, Western Blot, Quantitative RT-PCR, Gene Expression

Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 4. Inhibition of GLI1 alleviates pancreatic precancerous lesions by decreasing SLC25A1 expression in KrasG12D/þ mice. A, Experimental scheme for B–H. Sixty-day-old male and female ND-fed KrasG12D/þ mice were treated with TAM to induce KRASG12D expression in pancreatic acinar cells. These mice were fed HFD for 6 weeks and then randomly separated into two groups with one group fed HFD with vehicle (n ¼ 5) and another group of mice fed with HFD plus GANT61 treatment (intraperitoneally twice per week at 50 mg/kg/time; n ¼ 4) for 6 weeks. The mice were analyzed at the age of 150 days. B, Body weight of the KrasG12D/þ mice with ND (n ¼ 5), HFD (n ¼ 5), or HFD plus GANT61 treatment (n ¼ 4). Treatment started at the sixth week of feeding with HFD, as indicated by a red arrow. C, Gross images for pancreatic tissues of HFD-fed KrasG12D/þ mice with or without GANT61 treatment. The pancreatic cysts are indicated by red arrowswith dark edges. D, Representative H&E staining of the pancreata, Alcian blue staining of acidic mucins, Sirius red staining of collagens on pancreatic tissue sections, and coimmunofluorescence of pancreatic amylase and CK19 in HFD-fed KrasG12D/þ mice with or without GANT61 treatment (n ¼ 5; 200). E, Quantitation of Alcian blue and Sirius red staining levels in D. F, IHC staining of SLC25A1 (200), FASN (200), and ACSL1 (200) on pancreatic tissue sections of HFD-fed KrasG12D/þ mice with or without GANT61 treatment (n ¼ 5). G, Quantitation of SLC25A1, FASN, and ACSL1 levels in F. Results are expressed as mean SD and were statistically evaluated with a t test. , P < 0.01; , P < 0.001; , P < 0.0001. H, Western blot analysis of GLI1, SLC25A1, ACSL1, and FASN in the pancreata of HFD-fed KrasG12D/þ mice with or without GANT61 treatment. I, De novo FAS from glucose is decreased in PANC1 cells following GANT61 treatment. U-13C6-glucose metabolic tracing profile for methyl-palmitate (Me-C16:0) and methyl-stearate (Me-C18:0) chain elongation in PANC1 cells after treatment with either 0.13% DMSO (Control; n ¼ 3) or GANT61 (30 mmol/L, also dissolved in 0.13% DMSO; n ¼ 2) for 24 hours. Free FAs were extracted by organic solvents, derivatized as FAMEs and analyzed by GC-MS. The intensity of the endogenous U-12C-Me-C16:0 (Mþ0) and U-12C-Me-C18:0 (Mþ0) and the metabolically labeled 13C-isotopomers of Me-C16:0 (Mþ2 Mþ16) and Me-18:0 (Mþ2 Mþ18) are indicated. The ratio of labeled over the total (unlabeled þ labeled) was calculated. Results are expressed as mean SEM and were statistically analyzed by the multiple unpaired Student t test with Welch correction. , P < 0.05.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Inhibition, Expressing, Staining, Quantitation Assay, Immunohistochemistry, Western Blot, Control, Gas Chromatography-Mass Spectrometry, Metabolic Labelling, Labeling

Journal: Cancer Research

Article Title: Oncogenic KRASG12D Reprograms Lipid Metabolism by Upregulating SLC25A1 to Drive Pancreatic Tumorigenesis

doi: 10.1158/0008-5472.can-22-2679

Figure Lengend Snippet: Figure 6. SLC25A1 inhibitor suppressed PDAC development in KrasG12D/þ mice under a long-term HFD challenge. A, Experimental scheme for B–D. Sixty-day-old ND-fed KrasG12D/þ mice were treated with TAM to induce KRASG12D expression in pancreatic acinar cells. These male and female mice were randomly separated into two groups, with one group of mice (n ¼ 5) fed with HFD for 12 weeks, and pancreatic tissue samples were collected at 150 days of age as indicated by the control group. Another group of mice was fed HFD for 12 weeks and then fed with HFD plus CTPI-2 treatment (intraperitoneally twice per week at 50 mg/kg/time; n ¼ 5) for additional 6 weeks as indicated by the CTPI-2 group. B, Gross images of HFD-fed KrasG12D/þ mice with or without CTPI-2 treatment. C, Representative histology shown by H&E staining, Alcian blue staining, and Sirius red staining on pancreatic tissue sections from the HFD-fed control group and CPTI-2 treatment group (200). D, Quantitation of Alcian blue and Sirius red staining levels in C. E, Hypothetical model of the KRAS-GLI1-SLC25A1 axis that regulates lipid metabolism. Oncogenic KRASG12D upregulates SLC25A1 expression by increasing GLI1 transcription. More SLC25A1 exports more citrate from mitochondria to the cytosol for FAS, leading to severe lipid accumulation. Thus, SLC25A1 acts as a nodal transporter to enhance lipid metabolism. Targeted inhibition of the KRASG12D-GLI1-SLC25A1 axis by GANT61 or CTPI-2 could suppress cytosolic citrate and FA accumulation under HFD, therefore hindering pancreatic tumorigenesis. Results are expressed as mean SD and were statistically evaluated with a t test. , P < 0.01; , P < 0.0001.

Article Snippet: The primary antibodies for Western blot analysis and IHC were purchased as follows: SLC25A1 (NBP293363, Novus), ACSL1 (NBP1-60016, Novus), GLI1 (NB600-600, Novus; sc515751, Santa Cruz Biotechnology), CPT1A (NBP159608, Novus), CPT2 (NBP2-67699, Novus), FASN (3180s, Cell Signaling Technology), vimentin (5741s, Cell Signaling Technology), aSMA (19245s, Cell Signaling Technology), phospho-SAPK/JNK (Thr183/Tyr185; 4668s, Cell Signaling Technology), phosphop44/42 MAPK (Erk1/2; Thr202/Tyr204; 4370s, Cell Signaling Technology), GAPDH (sc-32233, Santa Cruz Biotechnology), a-tubulin (sc-5286, Santa Cruz Biotechnology), b-actin (sc-47778, Santa Cruz Biotechnology; NB600-501, Novus), CK19 (Ab52625, Abcam), and amylase (sc-46657, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Staining, Quantitation Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: A Potent Antagonist of Smoothened in Hedgehog Signaling for Epilepsy

doi: 10.3390/ijms232314505

Figure Lengend Snippet: The expression of Gli1 stimulated with Shh-CM was suppressed by TT22. ( A ) Serum-starved NIH3T3 cells were treated with Shh-CM for 24 h, then analyzed for mRNA levels of Gli1. ( B , C ) Serum-starved NIH3T3 cells were induced by Shh-CM with DMSO (vehicle), with an indicated concentration of TT22, 1 μM LDE-225 (LDE-1) or 1 μM GDC-0449 (GDC-1) for 24 h. Cells were analyzed for mRNA levels ( B ) and the protein levels ( C ) of Gli1. ( D ) Quantitation of Gli1 protein levels normalized to Tubulin loading control measured the dose-dependent inhibition of the Hh pathway activity by TT22 upon Shh stimulation. All data are means ± SEM (Student’s t -test and one-way ANOVA). *** p < 0.001.

Article Snippet: Cell lysates were harvested and detected by Western blotting with the following primary antibodies: Gli1 (CST, 2534s, 1:1000, Boston, MA, USA); and Tubulin (CST, 3873 s, 1:2000).

Techniques: Expressing, Concentration Assay, Quantitation Assay, Control, Inhibition, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: A Potent Antagonist of Smoothened in Hedgehog Signaling for Epilepsy

doi: 10.3390/ijms232314505

Figure Lengend Snippet: The expression of Gli1 stimulated with SAG was suppressed by TT22. ( A ) Serum-starved NIH3T3 cells were treated with 100 nM SAG for 24 h, then analyzed for mRNA levels of Gli1. ( B , C ) Serum-starved NIH3T3 cells were induced by 10 −7 M (100 nM) SAG with DMSO (vehicle), with the indicated concentration of TT22, 1 μM LDE-225 (LDE-1) or 1 μM GDC-0449 (GDC-1) for 24 h. Cells were analyzed for mRNA levels ( B ) and the protein levels ( C ) of Gli1. ( D ) Quantitation of Gli1 protein levels normalized to Tubulin loading control measured the dose-dependent inhibition of the Hh pathway activity by TT22 upon SAG stimulation. All data are means ± SEM (Student’s t -test and one-way ANOVA). *** p < 0.001.

Article Snippet: Cell lysates were harvested and detected by Western blotting with the following primary antibodies: Gli1 (CST, 2534s, 1:1000, Boston, MA, USA); and Tubulin (CST, 3873 s, 1:2000).

Techniques: Expressing, Concentration Assay, Quantitation Assay, Control, Inhibition, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: A Potent Antagonist of Smoothened in Hedgehog Signaling for Epilepsy

doi: 10.3390/ijms232314505

Figure Lengend Snippet: Scheme of the inhibition of Smoothened antagonists on seizure-like activity. Antagonists TT22, GDC-0449, and LDE-225 can bind with a Smoothened receptor, repress its activity, and then inhibit the expression of a downstream Gli1 transcription factor, thus suppressing the abnormal seizure-like activity in hippocampal neurons.

Article Snippet: Cell lysates were harvested and detected by Western blotting with the following primary antibodies: Gli1 (CST, 2534s, 1:1000, Boston, MA, USA); and Tubulin (CST, 3873 s, 1:2000).

Techniques: Inhibition, Activity Assay, Expressing

Journal: Virology

Article Title: Epstein-Barr virus latent membrane protein 2A mediated activation of Sonic Hedgehog pathway induces HLA class Ia downregulation in gastric cancer cells.

doi: 10.1016/j.virol.2015.05.007

Figure Lengend Snippet: Fig. 2. EBV LMP2A activates the Sonic Hedgehog pathway. (A) Quantitative RT-PCR of Sonic Hedgehog pathway genes in SNU719, AGS-LMP2A and HEK-LMP2A relative to AGS and HEK293 control cells respectively. (B) Immunoblotting experiments to determine Gli1 and Gli3 levels in AGS Control, SNU719, AGS-LMP2A and HEK293 Control and HEK-LMP2A. Bar graph depicts the densitometry quantification of relative expression levels of Gli1 and Gli3. (C) Real Time PCR analysis of Sonic Hedgehog pathway genes upon siRNA mediated knockdown of LMP2A expression in SNU719 cells at 24, 48 and 72 h respectively. (D) Protein levels of Gli1 and Gli3 in SNU719 cellsþLMP2A siRNA (72 h). Gli1 expression in AGSþLMP2A siRNA (72 h) relative to AGS control cells. Results are represented as mean7s.e.m. of 3 individual experiments. (E) Quantitative Real Time PCR indicating Gli1 and Gli3 expression in AGS cells transfected LMP2A siRNA. scr siRNA indicates scrambled siRNA.

Article Snippet: Gli1 and Gli3 siRNAs were obtained from Santa Cruz (California, USA).

Techniques: Quantitative RT-PCR, Control, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Knockdown, Transfection

Journal: Virology

Article Title: Epstein-Barr virus latent membrane protein 2A mediated activation of Sonic Hedgehog pathway induces HLA class Ia downregulation in gastric cancer cells.

doi: 10.1016/j.virol.2015.05.007

Figure Lengend Snippet: Fig. 3. Decrease in HLA ABC expression upon LMP2A expression is via the Hedgehog pathway. (A) Effect of downstream modulator Gli1 upon inhibiting the Shh pathway in SNU719 cells using forskolin. Dose and time response of HLA ABC expression when SNU719 cells are treated with Forskolin compared to untreated cells. Flow cytometric analysis of SNU719 cells treated with Forskolin (30 μM, 48 h). (B) Gli1 expression in AGS-LMP2A cells upon inhibiting the Shh pathway using forskolin. Dose and time response of HLA ABC expression when AGS-LMP2A cells are treated with Forskolin compared to untreated cells. Flow cytometric analysis of AGS-LMP2A cells treated with Forskolin (30 μM, 48 h). (C) Effect of Forskolin on Hedgehog pathway gene Gli1 in a dose and time dependent manner in HEK-LMP2A. Dose and time response of HLA ABC expression when HEK-LMP2A cells are treated with Forskolin compared to untreated cells. Flow cytometric analysis of HEK-LMP2A cells treated with Forskolin (30 μM, 48 h). (D) Quantitative RT-PCR of HLA A, B and C expression of SNU719þForskolin relative to untreatedSNU719. (E) Quantitative RT-PCR of HLA A, B and C expression of AGS- LMP2AþForskolin relative to untreated AGS-LMP2A. (F) mRNA expression levels of HLA A, B and C in HEK-LMP2AþForskolin relative to untreated HEK-LMP2A. The mean values7standard error obtained from atleast three independent experiments are shown. *, ** and *** denote P value o0.05, o0.005 ando0.001 respectively.

Article Snippet: Gli1 and Gli3 siRNAs were obtained from Santa Cruz (California, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Virology

Article Title: Epstein-Barr virus latent membrane protein 2A mediated activation of Sonic Hedgehog pathway induces HLA class Ia downregulation in gastric cancer cells.

doi: 10.1016/j.virol.2015.05.007

Figure Lengend Snippet: Fig. 4. LMP2A dependent HLA ABC downregulation is manifested by Hedgehog downstream mediators. (A–C) HLA ABC expression upon treating SNU719 (A), AGSLMP2A (B) and HEK LMP2A (C) cells with GSI, PNU74654 and LY294002. (D) Time dependent response of HLA ABC expression when SNU719 cells are treated with 20 nM Gli1 siRNA relative to untreated cells. Flow cytometric analysis of SNU719 cells treated with Gli1 siRNA for 72 h. Quantitative RT-PCR of HLA A, B and C expression in SNU719þGli1 siRNA relative to untreated SNU719 cells. (E) Time dependent response of HLA ABC expression when AGS-LMP2A cells are treated with Gli1 siRNA relative to untreated cells. Flow cytometric analysis of AGS-LMP2A cells treated with Gli1 siRNA for 72 h. Quantitative RT-PCR of HLA A, B and C expression of AGS-LMP2AþGli1 siRNA relative to untreated AGS-LMP2A. (F) Time dependent response of HLA ABC expression when HEKþLMP2A cells are treated with 20 nM Gli1 siRNA relative to untreated cells. Flow cytometric analysis of HEK-LMP2A cells treated with Gli1 siRNA for 72 h. Quantitative RT-PCR of HLA A, B and C expression in HEK-LMP2AþGli1 siRNA relative to untreated HEK-LMP2A. Results are represented as mean7s.e.m. of 3 individual experiments.

Article Snippet: Gli1 and Gli3 siRNAs were obtained from Santa Cruz (California, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Virology

Article Title: Epstein-Barr virus latent membrane protein 2A mediated activation of Sonic Hedgehog pathway induces HLA class Ia downregulation in gastric cancer cells.

doi: 10.1016/j.virol.2015.05.007

Figure Lengend Snippet: Fig. 5. LMP2A dependent HLA ABC downregulation is Gli1 specific. (A–C) HLA Class Ia expression upon treating SNU719 (A), AGS LMP2A (B) and HEK-LMP2A (C) with Gli3 siRNA. (D–E) HLA ABC expression upon introduction of Gli1 cDNA into AGS (D) and HEK293 cells (E). Flow cytometric analysis of AGS (D) and HEK293 (E) cells when treated with Gli1 cDNA for 72 h. Transcript level expression of HLA-A, B and C mRNA in AGSþGli1 (D) and HEK293þGli1 (E) at 24, 48 and 72 h relative to their respective vector control cells. The mean values7standard error obtained from atleast three independent experiments are shown. *, ** and *** denote P value o0.05, o0.005 ando0.001 respectively.

Article Snippet: Gli1 and Gli3 siRNAs were obtained from Santa Cruz (California, USA).

Techniques: Expressing, Plasmid Preparation, Control

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) GLI1 mRNA levels according to TCGA data for patients with the seven deadliest cancers in the United States in 2014. The data are medians with the 5th and 95th percentiles and standard deviations (error bars).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques:

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) In vitro methylation assay with PRMT1 and wild-type (WT) or R597K-mutant Gli1. Left panel, Coomassie Blue staining. Right panel, fluorography.

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: In Vitro, Methylation, Mutagenesis, Staining

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) Left panel, Western blot analysis of meGli1 and total Gli1 in MIA PaCa-2 luciferase cells stably transfected with an empty vector (Vec), wild-type Gli1 (Gli1WT), or R597K-mutant Gli1 (Gli1RK). Right panel, mRNA expression levels, measured by quantitative real-time PCR, of Gli1 target genes in Vec-, Gli1WT (WT)-, and Gli1RK (RK)-transfected MIA PaCa-2 cells. Error bars represent SD (n = 3). *P < 0.05, **P < 0.01 (paired two-tailed Student’s t-test).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: Western Blot, Luciferase, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cancer research

Article Title: Oncogenic functions of Gli in pancreatic adenocarcinoma are supported by its PRMT1-mediated methylation

doi: 10.1158/0008-5472.CAN-16-0715

Figure Lengend Snippet: (A) Responses of MIA PaCa-2 stable clones to gemcitabine with or without PRMT1 depletion. shCtrl: control shRNA; shPRMT1: PRMT1 shRNA; Vec: MIA PaCa-2 stable clone with empty vector; WT: stable clone with wild-type Gli1; RK: stable clone with Gli1R597 mutant. Error bars represent SD (n = 4).

Article Snippet: To generate Gli1 knockout cells, AsPC-1 cells were co-transfected with Gli1 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, sc-400266) and Gli1 HDR plasmid (Santa Cruz Biotechnology, sc-400266-HDR).

Techniques: Clone Assay, Control, shRNA, Stable Transfection, Plasmid Preparation, Mutagenesis