Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Quantitative real-time PCR analysis of the GalNAc4S-6ST transcript and profile of sulfation pattern of CS in the LLC-4S6ST-shRNA cells. (a) Total RNA was extracted from the control- (mock) or GalNAc4S-6ST-shRNA/LLC cells, and cDNA was synthesized by reverse transcriptase. Real-time PCR was conducted using the cDNAs, Taq polymerase, and SYBER Green. The expression of GalNAc4S-6ST was individually normalized to that of G3pdh . The assay was performed at least twice in triplicate, and representative results are shown. Values represent the mean ± SD. * P < 0.005; ** P < 0.001 versus control by one-way ANOVA with Dunnett's adjustment. (b, c) Anion-exchange HPLC of disaccharides obtained from the digests of CS derived from control- and GalNAc4S-6ST-shRNA/LLC cells with a mixture of chondroitinases ABC and AC-II. The GAG-peptide preparations from LLC cells, which stably express control-shRNA (b) or 4S6ST-shRNA (c), were individually digested with a mixture of chondroitinases ABC and AC-II. Each digest was labeled with a fluorophore 2AB as detailed in and analyzed by anion-exchange HPLC on an amine-bound silica PA03 column using a linear gradient of NaH 2 PO 4 as indicated by the dashed lines. The eluate was monitored by fluorescence intensity with the excitation and emission wavelengths of 330 and 420 nm, respectively. The insets show magnified chromatograms (10-fold) around the elution position of ΔE units. The positions of the 2AB-derivatized authentic disaccharides are indicated by numbered arrows: 1: ΔO, ΔHexUA-GalNAc; 2: ΔC, ΔHexUA-GalNAc(6- O -sulfate); 3: ΔA, ΔHexUA-GalNAc(4- O -sulfate); 4: ΔD, ΔHexUA(2- O -sulfate)-GalNAc(6- O -sulfate); 5: ΔB, ΔHexUA(2- O -sulfate)-GalNAc(4- O -sulfate); 6: ΔE, ΔHexUA-GalNAc(4- O -, 6- O -disulfate); 7: ΔT, ΔHexUA(2- O -sulfate)-GalNAc(4- O -, 6- O -disulfate).

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, shRNA, Control, Synthesized, Reverse Transcription, Expressing, Derivative Assay, Stable Transfection, Labeling, Fluorescence

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Disaccharide composition of CS chains in the control- and 4S6ST-shRNA/LLC cells. The GAG-peptide preparation from each cell line was digested with a mixture of chondroitinases ABC and AC-II and analyzed by anion-exchange HPLC after labeling with a fluorophore 2AB as detailed in Section 2 .

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Control, Labeling

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the pulmonary metastasis of LLC cells. Control- or GalNAc4S-6ST-shRNA/LLC-cell suspensions of 1 × 10 6 cells in 200 μ L of DMEM were injected into a tail vein of C57BL/6J mice, and 21 days later, the number of tumor foci in the lungs was recorded. Six mice were used per group. Representative lungs from mice injected with the LLC cells stably expressing control- (mock) or GalNAc4S-6ST-shRNA ( 4S6ST-shRNA ) are shown in (a). The number of colonies (b) and the wet weight (c) of the lungs from mice injected with each LLC clone were measured. Data represent the mean ± S.D. for two independent experiments. * P < 0.001 versus control by one-way ANOVA with Dunnett's adjustment.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Control, shRNA, Injection, Stable Transfection, Expressing

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the adhesion of LLC cells. LLC cells stably expressing control-shRNA (filled bars) or GalNAc4S-6ST-shRNA (open bars) were seeded on laminin-, fibronectin-, or collagen type IV-precoated plastic cover slips for 1-2 h. The cells were stained with Diff-Quik, and the adherent cells were counted. Average values obtained from three clones of both control-shRNA (nos. 5, 10, and 14) and GalNAc4S-6ST-shRNA (nos. 7, 17, and 23) are shown, and the experiments were performed in triplicate. Error bars indicate ±S.D. of triplicate samples. * P < 0.05 versus control-shRNA by Student's t -test.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Stable Transfection, Expressing, Control, shRNA, Staining, Diff-Quik, Clone Assay

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the invasion and migration of LLC cells. LLC cells stably expressing control-shRNA (filled bars) or GalNAc4S-6ST-shRNA (open bars) were plated on BD BioCoat chambers in the absence of fetal bovine serum. Cell invasion and migration were measured with or without Matrigel (BD Biosciences), respectively, as described in . Effects of the knockdown of GalNAc4ST-6ST on the invasion (a) and migration (b) of the LLC cells are summarized. The data represent the mean values ± S.D. for three clones of both control-shRNA (nos. 5, 10, and 14) and GalNAc4S-6ST-shRNA (nos. 7, 17, and 23). Three independent experiments were performed, and the representative results are shown. * P < 0.05 versus control-shRNA by Student's t -test.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Migration, Stable Transfection, Expressing, Control, shRNA, Clone Assay

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the proliferation of LLC cells. LLC cells stably expressing control-shRNA (mock) or GalNAc4S-6ST-shRNA ( 4S6ST-shRNA ) were seeded on 96-well plates at 2 × 10 3 cells/well and incubated at 37°C for the assessed period. Cell numbers were measured every 24 h using TetraColor One reagent containing a tetrazolium and electronic carrier as described in . The data represent the mean ± S.D. for three clones of both control-shRNA (nos. 5, 10, and 14) and GalNAc4S-6ST-shRNA (nos. 7, 17, and 23). Three independent experiments were performed, and representative results are shown. * P < 0.001 versus control-shRNA by Student's t -test.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Stable Transfection, Expressing, Control, shRNA, Incubation, Clone Assay

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Quantitative real-time PCR analysis of the GalNAc4S-6ST transcript and profile of sulfation pattern of CS in the LLC-4S6ST-shRNA cells. (a) Total RNA was extracted from the control- (mock) or GalNAc4S-6ST-shRNA/LLC cells, and cDNA was synthesized by reverse transcriptase. Real-time PCR was conducted using the cDNAs, Taq polymerase, and SYBER Green. The expression of GalNAc4S-6ST was individually normalized to that of G3pdh . The assay was performed at least twice in triplicate, and representative results are shown. Values represent the mean ± SD. * P < 0.005; ** P < 0.001 versus control by one-way ANOVA with Dunnett's adjustment. (b, c) Anion-exchange HPLC of disaccharides obtained from the digests of CS derived from control- and GalNAc4S-6ST-shRNA/LLC cells with a mixture of chondroitinases ABC and AC-II. The GAG-peptide preparations from LLC cells, which stably express control-shRNA (b) or 4S6ST-shRNA (c), were individually digested with a mixture of chondroitinases ABC and AC-II. Each digest was labeled with a fluorophore 2AB as detailed in and analyzed by anion-exchange HPLC on an amine-bound silica PA03 column using a linear gradient of NaH 2 PO 4 as indicated by the dashed lines. The eluate was monitored by fluorescence intensity with the excitation and emission wavelengths of 330 and 420 nm, respectively. The insets show magnified chromatograms (10-fold) around the elution position of ΔE units. The positions of the 2AB-derivatized authentic disaccharides are indicated by numbered arrows: 1: ΔO, ΔHexUA-GalNAc; 2: ΔC, ΔHexUA-GalNAc(6- O -sulfate); 3: ΔA, ΔHexUA-GalNAc(4- O -sulfate); 4: ΔD, ΔHexUA(2- O -sulfate)-GalNAc(6- O -sulfate); 5: ΔB, ΔHexUA(2- O -sulfate)-GalNAc(4- O -sulfate); 6: ΔE, ΔHexUA-GalNAc(4- O -, 6- O -disulfate); 7: ΔT, ΔHexUA(2- O -sulfate)-GalNAc(4- O -, 6- O -disulfate).

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, shRNA, Control, Synthesized, Reverse Transcription, Expressing, Derivative Assay, Stable Transfection, Labeling, Fluorescence

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Disaccharide composition of CS chains in the control- and 4S6ST-shRNA/LLC cells. The GAG-peptide preparation from each cell line was digested with a mixture of chondroitinases ABC and AC-II and analyzed by anion-exchange HPLC after labeling with a fluorophore 2AB as detailed in Section 2 .

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Control, Labeling

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the pulmonary metastasis of LLC cells. Control- or GalNAc4S-6ST-shRNA/LLC-cell suspensions of 1 × 10 6 cells in 200 μ L of DMEM were injected into a tail vein of C57BL/6J mice, and 21 days later, the number of tumor foci in the lungs was recorded. Six mice were used per group. Representative lungs from mice injected with the LLC cells stably expressing control- (mock) or GalNAc4S-6ST-shRNA ( 4S6ST-shRNA ) are shown in (a). The number of colonies (b) and the wet weight (c) of the lungs from mice injected with each LLC clone were measured. Data represent the mean ± S.D. for two independent experiments. * P < 0.001 versus control by one-way ANOVA with Dunnett's adjustment.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Control, shRNA, Injection, Stable Transfection, Expressing

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the adhesion of LLC cells. LLC cells stably expressing control-shRNA (filled bars) or GalNAc4S-6ST-shRNA (open bars) were seeded on laminin-, fibronectin-, or collagen type IV-precoated plastic cover slips for 1-2 h. The cells were stained with Diff-Quik, and the adherent cells were counted. Average values obtained from three clones of both control-shRNA (nos. 5, 10, and 14) and GalNAc4S-6ST-shRNA (nos. 7, 17, and 23) are shown, and the experiments were performed in triplicate. Error bars indicate ±S.D. of triplicate samples. * P < 0.05 versus control-shRNA by Student's t -test.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Stable Transfection, Expressing, Control, shRNA, Staining, Diff-Quik, Clone Assay

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the invasion and migration of LLC cells. LLC cells stably expressing control-shRNA (filled bars) or GalNAc4S-6ST-shRNA (open bars) were plated on BD BioCoat chambers in the absence of fetal bovine serum. Cell invasion and migration were measured with or without Matrigel (BD Biosciences), respectively, as described in . Effects of the knockdown of GalNAc4ST-6ST on the invasion (a) and migration (b) of the LLC cells are summarized. The data represent the mean values ± S.D. for three clones of both control-shRNA (nos. 5, 10, and 14) and GalNAc4S-6ST-shRNA (nos. 7, 17, and 23). Three independent experiments were performed, and the representative results are shown. * P < 0.05 versus control-shRNA by Student's t -test.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Migration, Stable Transfection, Expressing, Control, shRNA, Clone Assay

Journal: BioMed Research International

Article Title: Expression of N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Involved in Chondroitin Sulfate Synthesis Is Responsible for Pulmonary Metastasis

doi: 10.1155/2013/656319

Figure Lengend Snippet: Effects of the knockdown of GalNAc4S-6ST on the proliferation of LLC cells. LLC cells stably expressing control-shRNA (mock) or GalNAc4S-6ST-shRNA ( 4S6ST-shRNA ) were seeded on 96-well plates at 2 × 10 3 cells/well and incubated at 37°C for the assessed period. Cell numbers were measured every 24 h using TetraColor One reagent containing a tetrazolium and electronic carrier as described in . The data represent the mean ± S.D. for three clones of both control-shRNA (nos. 5, 10, and 14) and GalNAc4S-6ST-shRNA (nos. 7, 17, and 23). Three independent experiments were performed, and representative results are shown. * P < 0.001 versus control-shRNA by Student's t -test.

Article Snippet: Short hairpin RNA- (shRNA-) expressing plasmids (cat no.: sc-145317-SH) specific to mouse GalNAc4S-6ST, which target 5′-CUACAAUGUGGGAUAACAA-3′, 5′-CAAGACACCCUUAGAAUGU-3′, and 5′-GAACACUCGUGCUUAUACU-3′ and scrambled nucleotide sequence-containing control-shRNA plasmids (cat no.: sc-108060), shRNA transfection reagent, and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnologies, Inc (Santa Cruz, CA, USA).

Techniques: Knockdown, Stable Transfection, Expressing, Control, shRNA, Incubation, Clone Assay

Journal: Theranostics

Article Title: A novel miR-1291-ERRα-CPT1C axis modulates tumor cell proliferation, metabolism and tumorigenesis

doi: 10.7150/thno.44877

Figure Lengend Snippet: miR-1291 inhibits cancer cell proliferation and metabolism. (A) A WST-8 assay was performed to examine the viability of PANC-1 and MDA-MB-231 cells after overexpression of miR-1291. BrdU activity was used to measure cell proliferation capacity after treated with miR-1291 plasmid. Data are mean ± SD ( n = 5). (B) For colony formation assays, the cells were stained with Diff-Quik after being cultured for an additional 14 days. (C) The cell cycle was determined by flow cytometry after transfection with miR-1291 and immunoblot analysis of cell cycle-related proteins, such as cyclin A/D/E after transfection with miR-1291. Data are mean ± SD ( n = 3). (D) ATP production in miR-1291-transfected cells. Data are mean ± SD ( n = 5). (E) Intracellular accumulation of ROS in two cell lines. Data are mean ± SD ( n = 5). (F) RT-qPCR analysis to determine the expression of the mitochondriogenesis-related NRF1, PGC-1A, TFAM, and CYBA mRNAs . Data are mean ± SD ( n = 6). (G) Glycolysis inhibition test with 2-deoxyglucose and a glucose deprivation test with glucose to measure the anti-metabolic stress ability of cells. Data are mean ± SD ( n = 5).

Article Snippet: The miR-1291 and ERRα overexpression plasmids vectors were transfected at a concentration of 1 μg/10 6 cells using Mega DNA Transfection Reagent (Origene, USA) with the reduced serum medium Opti-MEM (Gibco, USA).

Techniques: Over Expression, Activity Assay, Plasmid Preparation, Staining, Diff-Quik, Cell Culture, Flow Cytometry, Transfection, Western Blot, Quantitative RT-PCR, Expressing, Inhibition

Journal: Theranostics

Article Title: A novel miR-1291-ERRα-CPT1C axis modulates tumor cell proliferation, metabolism and tumorigenesis

doi: 10.7150/thno.44877

Figure Lengend Snippet: miR-1291 indirectly regulates CPT1C. (A) Expression of CPT1C mRNA in PANC-1 and MDA-MB-231 cells after transfection with miR-1291. Data are mean ± SD ( n = 6). (B) Western blot assay was used to detect expression of CPT1C after transfection with miR-1291. Band intensity was evaluated by Quantity one software. Data are mean ± SD ( n = 3). (C) Luciferase reporter gene assays were conducted in HEK-293T cells treated with different CPT1C reporter plasmids to define the impact of miR-1291. ST-miR1291-PANC-1 and ST-miR1291-231 cell lines were also used to verify the effect of miR-1291 on luciferase activities of the CPTIC promoters. Data are mean ± SD ( n = 5).

Article Snippet: The miR-1291 and ERRα overexpression plasmids vectors were transfected at a concentration of 1 μg/10 6 cells using Mega DNA Transfection Reagent (Origene, USA) with the reduced serum medium Opti-MEM (Gibco, USA).

Techniques: Expressing, Transfection, Western Blot, Software, Luciferase

Journal: Theranostics

Article Title: A novel miR-1291-ERRα-CPT1C axis modulates tumor cell proliferation, metabolism and tumorigenesis

doi: 10.7150/thno.44877

Figure Lengend Snippet: miR-1291 targets the ERRα pathway. (A) The Venn diagram displayed the overlaps between four different gene sets. (B) Levels of ESRRA and PGC-1A mRNAs in PANC-1 and MDA-MB-231 cells after transfection with miR-1291. The data are the mean ± SD ( n = 6). (C) Western blot analysis of ERRα after transfection with miR-1291. Data are mean ± SD ( n = 3). (D) MDA-MB-231 cell lines were stained for ERRα. (E) ERRα target gene mRNA levels measured under the same conditions described above. Data are mean ± SD ( n = 6). (F) Bioinformatics analysis revealing two putative MRE sites for miR-1291 within the 3'UTR of ESRRA transcript. The seed sequence of miR-1291 is underlined. ESRRA 3'UTR luciferase reporter activities were determined. Data are mean ± SD ( n = 5). (G) The sequence of ESRRA 3'UTR bearing the mutated MRE sites is shown. The ESRRA 3'UTR luciferase reporter activities were detected with two ERRα -Reporter-MUT plasmids. Data are mean ± SD ( n = 5).

Article Snippet: The miR-1291 and ERRα overexpression plasmids vectors were transfected at a concentration of 1 μg/10 6 cells using Mega DNA Transfection Reagent (Origene, USA) with the reduced serum medium Opti-MEM (Gibco, USA).

Techniques: Transfection, Western Blot, Staining, Sequencing, Luciferase

Journal: Theranostics

Article Title: A novel miR-1291-ERRα-CPT1C axis modulates tumor cell proliferation, metabolism and tumorigenesis

doi: 10.7150/thno.44877

Figure Lengend Snippet: ERRα modulates cancer cell proliferation and metabolism. (A) WST-8 and BrdU assays were performed to examine the viability and proliferation capacity of PANC-1 and MDA-MB-231 cells after modulation of ESRRA levels by siRNA and expression plasmid. The data are the mean ± SD ( n = 5). (B) After transfection with the ERRα expression plasmid and siRNA ERRα, cells were cultured for an additional two weeks and stained with Diff-Quik to determine colony formation capacity. (C) The cell cycle was determined by flow cytometry after the transfection of ERRα siRNA. The protein levels of cell cycle-related proteins, such as cyclin A/D/E were determined by immunoblot analysis after transfection with ERRα siRNA. Data are mean ± SD ( n = 3). (D) ATP production in both high ERRα and low ERRα cells was detected. Data are mean ± SD ( n = 5). (E) The intracellular accumulation of ROS in two cell lines was examined following the depletion of ERRα. Data are mean ± SD ( n = 5). (F) Expression of mitochondriogenesis-related mRNAs NRF1, PGC-1A, TFAM , and CYBA. Data are mean ± SD ( n = 6). (G) A glycolysis inhibition test with 2-deoxyglucose and a glucose deprivation test with glucose were performed to measure the anti-metabolic stress ability of tumor cells. Data are mean ± SD ( n = 5).

Article Snippet: The miR-1291 and ERRα overexpression plasmids vectors were transfected at a concentration of 1 μg/10 6 cells using Mega DNA Transfection Reagent (Origene, USA) with the reduced serum medium Opti-MEM (Gibco, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Staining, Diff-Quik, Flow Cytometry, Western Blot, Inhibition