Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH inhibits the proliferation and induces apoptosis of NP cells. ( A ) The expression levels of circITCH were measured by qPCR in the NP tissues of IDD patients (n=90) and normal cases (n=90). ( B – F ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( B ) The expression levels of circITCH were examined by qPCR in the cells. ( C , D ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( E ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( F ) The expression of Bax, caspase3, cleaved caspase3 (c-caspase3), caspase9, and cleaved caspase9 (c-caspase9) was measured by Western blot analysis. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH promotes ECM degradation of degenerative NP cells. ( A – E ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( A – D ) The mRNA expression of collagen II ( A ), aggrecan ( B ), MMP13 ( C ), and ADAMTS4 ( D ) was measured by qPCR in the cells. ( E ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was tested by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Infection, shRNA, Control, Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH serves as a miR-17-5p sponge in NP cells. ( A ) The potential interaction between circITCH and miR-17-5p was identified by the bioinformatic analysis using ENCORI ( http://starbase.sysu.edu.cn/index.php ). ( B , C ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type circITCH (circITCH WT) and circITCH with the miR-17-5p-binding site mutant (circITCH MUT) were determined by luciferase reporter gene assays in the cells. ( D ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. The expression of miR-17-5p was analyzed by qPCR in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Control, Expressing, Luciferase, Binding Assay, Mutagenesis, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH activates Wnt/β-catenin signaling by targeting miR-17-5p/SOX4 axis. ( A ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and miR-17-5p inhibitor. The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( B , C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, lentiviral plasmids carrying circITCH shRNA, pcDNA3.1-SOX4 overexpression vector, and miR-17-5p mimic, or lentiviral plasmids carrying circITCH shRNA and LiCl. The expression of Wnt1, β-catenin, c-Myc, Cyclin D1, and β-actin was analyzed by Western blot analysis in the cells. The results of Western blot analysis were quantified by ImageJ software. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Expressing, Western Blot, Over Expression, Plasmid Preparation, Software

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH contributes to ECM degradation of degenerative NP cells by modulating miR-17-5p/SOX4/Wnt/β-catenin signaling. ( A – C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, miR-17-5p inhibitor, or LiCl. ( A ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( B ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( C ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, Control, shRNA, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: Primer used for RT-qPCR

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques:

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: DNMT3B upregulation is responsible for the hypermethylation of the MYH11 promoter. A , the expression of DNMT3A and DNMT3B in GC was queried in StarBase Pan-cancer platform; B , the enrichment ability of DNMT3A and DNMT3B on MYH11 promoter examined by ChIP-qPCR; C , DNMT3B mRNA expression in GC tumor tissues and their adjacent tissues by RT-qPCR; D , correlation of DNMT3B expression with MYH11 promoter methylation levels in tumor tissues analyzed by Pearson’s correlation analysis (r = 0.623, p < 0.001); E , correlation of DNMT3B expression with MYH11 expression in tumor tissues (r = − 0.609, p < 0.001); F , transfection efficiency of oe-DNMT3B in GC cells by RT-qPCR; G , the effects of DNMT3B overexpression on the methylation level of MYH11 promoter examined by qMSP; H , effects of DNMT3B overexpression on MYH11 expression by RT-qPCR. Each assay was performed at least three times. Statistical significance was analyzed by paired t test (panel C) or two-way ANOVA (panels B, F, G and H) and Tukey’s multiple range tests

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Expressing, Quantitative RT-PCR, Methylation, Transfection, Over Expression

Journal: BMC Cancer

Article Title: Interaction between DNMT3B and MYH11 via hypermethylation regulates gastric cancer progression

doi: 10.1186/s12885-021-08653-3

Figure Lengend Snippet: Mechanism diagram. Overexpression of DNMT3B in GC inhibited MYH11 expression by promoting methylation of the MYH11 promoter, thereby attenuating the repressive effect of MYH11 on TNFRSF14 transcriptional activity and promoting GC progression

Article Snippet: The overexpressed (oe) DNA plasmids oe-MYH11, oe-TNFRSF14, oe-DNMT3B and control oe-negative control (NC) used for cell transfection were from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Over Expression, Expressing, Methylation, Activity Assay

Journal: Oncology Reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Effect of KLF5 on cell growth in vitro . (A) Knockdown and overexpression efficacy of KLF5 verified by western blot analysis. (B) Cell proliferation of SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2 cells was evaluated using a CCK-8 assay (upper panel); Cell proliferation of B-CPAP/NC and B-CPAP/KLF5 cells was determined using a CCK-8 assay (lower panel). (C) Anchorage-independent growth assay in SW579 and B-CPAP cells. Knockdown of KLF5 inhibited the anchorage-independent growth of SW579 cells while the overexpression of KLF5 promoted the anchorage-independent growth of B-CPAP cells. KLF5, Kruppel-like factor 5; scrRNA, scrambled RNA; si-, small interfering RNA; NC, negative control; OD, optical density; CCK-8, Cell Counting Kit-8.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5′-CGATTACCCTGGTTGCACA-3′) or shKLF5#2 (target sequence: 5′-AAGCTCACCTGAGGACTCA-3′) were established.

Techniques: In Vitro, Over Expression, Western Blot, CCK-8 Assay, Growth Assay, Small Interfering RNA, Negative Control, Cell Counting

Journal: Oncology Reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: KLF5 promoted the invasive and metastatic potential of thyroid cancer cells in vitro . (A) Cell migration/invasion of SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2 was analyzed using a Transwell assay. (B) Cell migration/invasion of B-CPAP/NC and B-CPAP/KLF5 cells was assessed using a Transwell assay. (C) Western blot analysis of lysates from SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2 cells using anti-E-cadherin, anti-vimentin, anti-Twist1 and anti-fibronectin and (D) the western blot analysis results quantified. (E) Immunostaining of phalloidin (F-actin) in SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2 cells. (F) Western blot analysis of lysates from B-CPAP/NC and B-CPAP/KLF5 cells using the indicated antibodies and (G) the western blot analysis results quantified. (H) Immunostaining of phalloidin (F-actin) in B-CPAP/NC and B-CPAP/KLF5 cells. KLF5, Kruppel-like factor 5; scrRNA, scrambled RNA; si-, small interfering RNA; NC, negative control; Twist1, Twist family BHLH transcription factor 1.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5′-CGATTACCCTGGTTGCACA-3′) or shKLF5#2 (target sequence: 5′-AAGCTCACCTGAGGACTCA-3′) were established.

Techniques: In Vitro, Migration, Transwell Assay, Western Blot, Immunostaining, Small Interfering RNA, Negative Control

Journal: Oncology Reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Effect of KLF5 on the ERK pathway and the AKT pathway. Western blot analysis of p-ERK, ERK, p-AKT and AKT in SW579 cells (SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2) and B-CPAP cells (B-CPAP/NC and B-CPAP/KLF5). KLF5, Kruppel-like factor 5; scrRNA, scrambled RNA; si-, small interfering RNA; NC, negative control; AKT, protein kinase B; p-, phosphorylated; ERK, extracellular-regulated kinase; NS, not significant.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5′-CGATTACCCTGGTTGCACA-3′) or shKLF5#2 (target sequence: 5′-AAGCTCACCTGAGGACTCA-3′) were established.

Techniques: Western Blot, Small Interfering RNA, Negative Control

Journal: Oncology Reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: (A) Western blotting of p-IκB-α, p-IKK-β and nuclear NF-κB p65 in SW579 cells (SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2, left panel) and B-CPAP cells (B-CPAP/NC and B-CPAP/KLF5, right panel); and p84 was used as a nuclear marker. (B) Quantified western blotting results. (C) Immunofluorescent staining of NF-κB p65 in SW579/scrRNA, SW579/siKLF5#1, SW579/siKLF5#2, B-CPAP/NC and B-CPAP/KLF5 cells. NF-κB, nuclear factor-κB; p-, phosphorylated-; IKK-β, inhibitor of nuclear factor κB kinase subunit β; IκB-α, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α; scrRNA, scrambled RNA; si, small interfering RNA; NC, negative control; KLF5, Kruppel-like factor 5.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5′-CGATTACCCTGGTTGCACA-3′) or shKLF5#2 (target sequence: 5′-AAGCTCACCTGAGGACTCA-3′) were established.

Techniques: Western Blot, Marker, Staining, Small Interfering RNA, Negative Control

Journal: Oncology Reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: KLF5 promoted the in vivo tumor growth and metastasis of thyroid cancer cells. (A) Knockdown efficacy of KLF5 by two shRNAs was confirmed by western blotting. (B) SW579/shNC, SW579/shKLF5#1 and SW579/shKLF5#2 cells were subcutaneously injected into the right flanks of nude mice and (C) the growth curve of tumor volumes was obtained. (D) Mean weight of the tumors from the nude mice of the indicated groups. (E) Representative images of the immunohistochemistry analysis of KLF5, E-cadherin and ki67. (F) B-CPAP/NC and B-CPAP/KLF5 cells were injected intravenously into the tail vein of mice. Representative H&E staining results of lungs are presented. (G) Number of lung metastatic foci in the B-CPAP/NC and B-CPAP/KLF5 groups (5 mice per group). KLF5, Kruppel-like factor 5; shRNA/sh, short hairpin RNA; NC, negative control; H&E, hemotoxylin and eosin.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5′-CGATTACCCTGGTTGCACA-3′) or shKLF5#2 (target sequence: 5′-AAGCTCACCTGAGGACTCA-3′) were established.

Techniques: In Vivo, Western Blot, Injection, Immunohistochemistry, Staining, shRNA, Negative Control

Journal: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Schematic of DECR1 function in fatty acid (FA) β-oxidation. In order to translocate FAs into the mitochondria, CPT1 converts long-chain acyl-CoA species to their corresponding long-chain acylcarnitine species. This is followed by a dehydrogenation step mediated by acyl CoA dehydrogenase (ACAD) to generate trans-2-enoyl-CoA, the only intermediate that can be processed by downstream enzymes in the β-oxidation process. Many FAs have unsaturated bonds either on an odd-numbered carbon or in the cis-configuration, resulting in the generation of enoyl-CoA intermediates that cannot be directly processed via the downstream β-oxidation enzymes. These FAs require the activity of 3 auxiliary enzymes, ECI1, ECH1 and DECR1 in order to form trans-2-enoyl-CoA before undergoing β-oxidation. DECR1 catalyzes the conversion of either 2-trans,4-cis-dienoyl or 2-trans,4-trans-dienoyl-CoA to 3-trans-enoyl-CoA. A complete cycle of β-oxidation results in the release of the first two carbon units as acetyl-CoA, and a fatty-acyl-CoA minus two carbons. The acetyl-CoA enters the TCA cycle to produce energy (ATP). The shortened fatty-acyl-CoA is processed again starting with the ACADs to form trans-2-enoyl-CoA either directly or with the aid of the auxiliary enzymes depending on the presence of double bonds. This process continues until all carbons in the fatty acid chain are turned into acetyl-CoA. (B) DECR1 protein expression after 72 hours or 96 hours siRNA transfection. Densitometry quantification of relative DECR1 protein expression was normalized to the HSP90 internal control. (C) Linoleic acid level in LNCaP cells quantified in following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. (D) Relative quantities of the C10:2 acylcarnitine species in LNCaP cell conditioned medium (left) or cell lysates (right) (n=3). (E) Quantification of ATP levels in LNCaP cell lysates. LNCaP cells were transfected with DECR1 siRNAs for 48 hours and then starved in no-glucose medium and treated with the lipolysis inhibitor DEUP (100µM) in the presence (BSA-LA) or absence (BSA) of the PUFA linoleic acid for 48 hours before measuring ATP levels. ( F) Oxygen consumption rate (OCR) was assessed in LNCaP cells supplemented with the PUFA linoleic acid (LA) or (G) the saturated fatty acid palmitic acid (PA). Each data point represents an OCR measurement. ATP production, maximal mitochondrial respiration and mitochondrial spare capacity were assessed. (H) Extracellular acidification rate (ECAR) was assessed in LNCaP cells. Each data point represents an ECAR measurement. For experiments (F-H) LNCaP cells were transfected with DECR1 siRNAs for 72 hours, then starved in substrate limited medium for 24 hours; the assay was run in FAO assay medium. (I & J) Metabolites were quantified in LNCaP cells following 96 hours DECR1 knockdown using GC QQQ targeted metabolomics. Data in bar graphs are represented as the mean ± s.e.m (n=3). Statistical analysis was performed using two-tailed Student’s t -test: * p <0.05, ** p <0.01 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Activity Assay, Expressing, Transfection, Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.1101/865626

Figure Lengend Snippet: (A) Cell viability after DECR1 knockdown in non-malignant PNT1 prostate cells; hormone-responsive PCa cell lines (LNCaP and VCaP); castrate-resistant V16D and 22RV1 cell lines and enzalutamide-resistant MR94F cells cultured in full serum media. (B) Cell viability of stable DECR1-overexpressed LNCaP cells cultured in full serum media. Cell viability and cell death were measured using trypan blue exclusion following 96 hours DECR1 knockdown. Percentages are represented relative to the control siRNA; n = 3 independent experiments per cell line. (C) Clonogenic cell survival of LNCaP cells were assessed using colony formation assay. Stable DECR1-overexpressed cells or (D) stable DECR1 knockdown was achieved using two different short hairpin (sh) vectors and DECR1 expression was confirmed using western blot. Cells were cultured for 2 weeks, washed with PBS, fixed with paraformaldehyde and stained with 1% crystal violet for 30 minutes. Colonies with more than 50 cells were counted manually; data shown is representative of n = 2 independent experiments. (E) LNCaP and 22RV1 cell growth in 3D spheres. Spheroids were prepared using the hang drop assay following 48 hours DECR1 knockdown. Spheroid volumes were determined after five days of culturing the cells in 20 µl drops; at least 25 spheres per cell line were assessed using the ReViSP software, n = 3 independent experiments per cell line. (F) LNCaP, 22RV1 and MR49F cell migration and (G) 22RV1 cell invasion were assessed using transwell migration/invasion assay. Cells were transfected with DECR1 siRNA or control siRNA for 48 hours. Equal number of cells were transferred to the upper inserts in serum free medium; lower chambers were filled with medium containing 5% serum as a chemoattractant. Plates were incubated for a further 48 hours. Migrated/invaded cells on the lower face of the inserts were washed with PBS, fixed with paraformaldehyde, stained with 1% crystal violet for 30 minutes, and counted manually; data shown is representative of n = 3 independent experiments. (H) Violin plots of mKi67 and DECR1 mRNA expression in LNCaP tumors (n = 5 mice, shControl; n = 4 mice, shDECR1). (I) Representative KI67 IHC staining of LNCaP tumors. Scale bar, 100µm. Data in bar graphs are represented as the mean ± s.e.m. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test: * p <0.05, ** p <0.01, *** p <0.001 and **** p <0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc. (San Diego, CA, USA) according to the manufacturer’s protocol.

Techniques: Knockdown, Cell Culture, Control, Colony Assay, Expressing, Western Blot, Staining, Software, Migration, Invasion Assay, Transfection, Incubation, Immunohistochemistry

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: The relative expression of BCAT1 in Ishikawa and HEC-1A cells. BCAT1 expression in Ishikawa and HEC-1A cells transfected with a blank control, shCtrl and shBCAT1. The y-axis indicates the relative expression of BCAT1 in each group tested using RT-qPCR.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR

Journal: International Journal of Clinical and Experimental Pathology

Article Title: BCAT1 promotes proliferation of endometrial cancer cells through reprogrammed BCAA metabolism

doi:

Figure Lengend Snippet: A. Heat map showing the changes in the concentrations (Z-score) of 26 amino acids and their derivatives after BCAT1 knockdown in HEC-1A cells. B. Intracellular amino acids and their derivative levels in shCtrl cells and shBCAT1 cells.

Article Snippet: Short Hairpin RNA (shRNA) - mediated BCAT1 knockdown in HEC-1A and Ishikawa cells To knockdown the expression of BCAT1, shRNAs targeting BCAT1 mRNA (shBCAT1) and a non-targeting negative control shRNA (shCtrl), which are expressed with lentivirus, were purchased from GenePharma (Shanghai, China).

Techniques: Knockdown