Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro suppresses NSCLC cell proliferation. (A) Andro inhibited H1975 cell survival in a dose-time-dependent manner, as measured by MTT assessment; (B) Andro inhibited H1975 cell clone formation in a dose-dependent manner, as measured through the colony-formation assay. Colony formation capacities were determined by crystal violet staining and the magnification was 40× under optical microscope. *, P<0.05; **, P<0.01; ***, P<0.001. Andro, andrographolide; NSCLC, non-small cell lung cancer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Colony Assay, Staining, Microscopy

Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro exposure promotes NSCLC cell apoptosis. (A) Andro facilitated H1975 cell apoptosis in a dose-dependent manner, as measured through flow cytometry; (B) Andro enhanced cleaved caspase 9, cleaved caspase 8 , and c leaved caspase 3 expression in H1975 cells in a dose-dependent manner, as measured by WB. *, P<0.05; **, P<0.01; ***, P<0.001. Andro, andrographolide; NSCLC, non-small cell lung cancer; WB, western blotting.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Flow Cytometry, Expressing, Western Blot

Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro inhibits NSCLC cell proliferation by activating the mitochondrial apoptosis pathway. (A) Andro weakened ΔΨm in H1975 cells, as measured by JC-1 fluorescence. Scale bar: 20 µm; (B) Andro inhibited mitochondrial cyto C and cytoplasmic Bak expressions, and promoted cytoplasmic cyto C and mitochondrial Bak expressions in H1975 cells in a time-dependent manner, as evaluated through WB; (C) Andro enhanced Bax and Bak and inhibited Bcl-2 expression in H1975 cells in a dose-dependent manner, as evaluated through WB; (D) Transfection with Bak-siRNA significantly inhibited Bak mRNA and protein expressions in H1975 cells, as determined by qRT-PCR and WB, respectively; (E) Bak downregulation promoted H1975 cell proliferation, as measured through CCK-8 assay; (F) Bak downregulation enhanced ΔΨm in H1975 cells, as determined by the JC-1 fluorescence experiment. Scale bar: 20 µm; (G) Bak downregulation markedly inhibited apoptotic executive proteins (cleaved caspase 9, cleaved caspase 8, and cleaved caspase 3) expression in H1975 cells, as measured by WB. *, P<0.05; **, P<0.01; ***, P<0.001. Andro, andrographolide; NSCLC, non-small cell lung cancer; ΔΨm, mitochondrial membrane potential; JC-1: mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide; cyto C, cytochrome C; Bcl-2, B-cell leukemia/lymphoma 2; Bak, Bcl-2-antagonist/killer (Bak); Bax, Bcl2-associated X; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mrna, messenger RNA; WB, western blotting; qRT-PCR, real-time quantitative reverse transcription-polymerase chain reaction; CCK-8, cell counting kit-8; OD, optical density; NC, negative control; si, small interfering.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Fluorescence, Expressing, Transfection, Quantitative RT-PCR, CCK-8 Assay, Membrane, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Cell Counting, Negative Control

Journal: Annals of Translational Medicine

Article Title: Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism

doi: 10.21037/atm-21-5975

Figure Lengend Snippet: Andro suppresses NSCLC cell proliferation by inducing the reprogramming of glucose metabolism. (A) Andro reduced the expression of PKM2, LDHA , and GLUT1 genes, and stimulated the expression of PEPCK1, FBP1 , and PFK genes in H1975 cells, as measured by qRT-PCR; (B) Andro inhibited glucose uptake, lactate production, and intracellular ATP synthesis in H1975 cells; (C) FBP1 -siRNA significantly inhibited FBP1 expression at both the mRNA and protein levels in H1975 cells, as measured by qRT-PCR and WB; (D) in H1975 cells, FBP1 downregulation promoted glucose uptake, lactate production, intracellular ATP synthesis; (E) FBP1 downregulation promoted H1975 cell proliferation, as evaluated through the CCK-8 assay; (F) FBP1 downregulation inhibited cleaved caspase 9, cleaved caspase 8, and cleaved caspase 3 gene expression, as evaluated through WB. *, P<0.05; **, P<0.01. Andro, andrographolide; NSCLC, non-small cell lung cancer; PKM2 , pyruvate kinase M2; LDHA , lactate dehydrogenase A; GLUT1 , glucose transporter 1; PEPCK1 , phosphoenolpyruvate carboxykinase 1; FBP1 , fructose-1,6-bisphosphatase 1; PFK , phosphofructokinase; GAPDH , glyceraldehyde-3-phosphate dehydrogenase; ATP, adenosine triphosphate; qRT-PCR, real-time quantitative reverse transcription-polymerase chain reaction; Mrna, messenger RNA; WB, western blotting; CCK-8, cell counting kit-8; OD, optical density; NC, negative control; si, small interfering.

Article Snippet: For the knock-down of B-cell leukemia/lymphoma 2 ( Bcl-2 )-antagonist/killer ( Bak ) or fructose-1,6-bisphosphatase ( FBP1 ) expression in H1975 cells, negative control (NC) (non-silencing control)-small interfering RNA (si-RNA) (NC-siRNA) and Bak targeting siRNA ( Bak -siRNA) (50 nM) were acquired from Qiagen and transfected into the above target cells using the RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s guidelines.

Techniques: Expressing, Quantitative RT-PCR, CCK-8 Assay, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Cell Counting, Negative Control

Journal: Oncology Letters

Article Title: Inhibition of lung tumor growth in nude mice by siRNA CD31 targeting PECAM-1

doi: 10.3892/ol.2014.2091

Figure Lengend Snippet: siRNA CD31 -RNAi-mate complexes successfully transfected into EOMA cells. (A) Bright fluorescence emitted from the EOMA cells transfected by the FAM-labeled siRNA CD31 . (B) The same EOMA cells observed by optical microscope. siRNA, small interfering RNA; CD, cluster of differentiation; EOMA, murine hemangioendothelioma.

Article Snippet: The 2′-O-methyl-modified siRNA CD31 molecules used in the present study are described in . siRNA CD31 , 3′-fluorescein amidite (FAM) fluorescence-labeled siRNA CD31 (siRNA CD31 -FAM; described in ), stable negative control RNA (SNC; described in ) and RNAi-mate were all synthesized by GenePharma Co., Ltd. (Shanghai, China).

Techniques: Transfection, Fluorescence, Labeling, Microscopy, Small Interfering RNA

Journal: Oncology Letters

Article Title: Inhibition of lung tumor growth in nude mice by siRNA CD31 targeting PECAM-1

doi: 10.3892/ol.2014.2091

Figure Lengend Snippet: Results of the MTT assay for the inhibition rates of EOMA proliferation showing that siRNA CD31 and siRNA CD31 -FAM effectively inhibit the proliferation of EOMA cells using RNAi-mate as a carrier (all P<0.01 vs. SNC). The inhibition rates of EOMA proliferation are not impaired for the fluorescence FAM-labeled siRNA CD31 in the EOMA cells treated by FAM-labeled siRNA CD31 -FAM-RNAi-mate (i.e., the siRNA CD31 -FAM group) (P>0.05 vs. siRNA CD31 ). The wells containing Opti-MEM were used as control wells. Each assay was performed in triplicate and was independently repeated three times. The inhibition rate of EOMA proliferation was calculated with the formula: Inhibition rate of proliferation (%) = (1 - OD experimental wells / OD of control wells) × 100. siRNA, small interfering RNA; CD31, cluster of differentiation 31; PECAM-1, platelet endothelial cell molecule 1; EOMA, murine hemangioendothelioma; RNAi, RNA interference; SNC, stable negative control; MEM, mimimum essential medium; OD, optical density. * P<0.01 vs. SNC.

Article Snippet: The 2′-O-methyl-modified siRNA CD31 molecules used in the present study are described in . siRNA CD31 , 3′-fluorescein amidite (FAM) fluorescence-labeled siRNA CD31 (siRNA CD31 -FAM; described in ), stable negative control RNA (SNC; described in ) and RNAi-mate were all synthesized by GenePharma Co., Ltd. (Shanghai, China).

Techniques: MTT Assay, Inhibition, Fluorescence, Labeling, Control, Small Interfering RNA, Negative Control

Journal: Oncology Letters

Article Title: Inhibition of lung tumor growth in nude mice by siRNA CD31 targeting PECAM-1

doi: 10.3892/ol.2014.2091

Figure Lengend Snippet: In vitro siRNA CD31 downregulates the expression of PECAM-1 mRNA and protein in EOMA cells. RT-PCR analysis of (A) PECAM-1 mRNA and (B) GADPH mRNA expression was used as an internal control. Western blot analysis of (C) PECAM-1 protein and (D) GADPH protein was used as an internal control. (E) Bar diagram showing the relative quantitative values of PECAM-1 mRNA and protein determined with RT-PCR and western blot analysis, respectively. siRNA CD31 and siRNA CD31 -FAM, with RNAi-mate as a carrier, downregulated the expression of PECAM-1 mRNA and protein compared with EOMA cells treated with SNC and Opti-MEM (i.e., control group) (all P<0.01 vs. SNC or control) and the effects were not weakened for fluorescence FAM-labeled siRNA CD31 (P>0.05, siRNA CD31 vs. siRNA CD31 -FAM). There was no significant difference between the SNC and control groups (P>0.05). The relative quantification value (RQ) of PECAM-1 mRNA (protein) was calculated according to the following equation: RQ PECAM-1 RNA (protein) = IOD PECAM-1 mRNA (protein) / IOD GADPH mRNA (protein). The RT-PCR and western blot analysis data shown were obtained from assays performed in triplicate and independently repeated three times. siRNA, small interfering RNA; PECAM-1, platelet endothelial adhesion molecule 1; CD31, cluster of differentiation 31; EOMA, murine hemangioendothelioma; RT-PCR, reverse transcription polymerase chain reaction; RNAi, RNA interference; SNC, stable negative control; MEM, mimimum essential medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IOD, integrated optical density. M, marker; lane 1, siRNA CD31 ; lane 2, siRNA CD31 -FAM; lane 3, SNC; lane 4, Opti-MEM used as control. Error bars shows the standard error of the mean. * P<0.01 vs. control; # P<0.01 vs. SNC.

Article Snippet: The 2′-O-methyl-modified siRNA CD31 molecules used in the present study are described in . siRNA CD31 , 3′-fluorescein amidite (FAM) fluorescence-labeled siRNA CD31 (siRNA CD31 -FAM; described in ), stable negative control RNA (SNC; described in ) and RNAi-mate were all synthesized by GenePharma Co., Ltd. (Shanghai, China).

Techniques: In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Fluorescence, Labeling, Quantitative Proteomics, Small Interfering RNA, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Marker

Journal: Oncology Letters

Article Title: Inhibition of lung tumor growth in nude mice by siRNA CD31 targeting PECAM-1

doi: 10.3892/ol.2014.2091

Figure Lengend Snippet: Growth of tumor xenografts of nude mice treated by the tail vein injection of siRNA CD31 -RNAi-mate complexes (siRNA CD31 group) is slower than those treated with saline (i.e., control). The tumor xenograft volumes in the nude mice treated with siRNA CD31 -RNAi-mate complexes were smaller than those in the control nude mice from day 4, and the deviation of tumor volumes (DV) increased, continuing to day 10. siRNA, small interfering RNA; CD31, cluster of differentiation 31; RNAi, RNA interference.

Article Snippet: The 2′-O-methyl-modified siRNA CD31 molecules used in the present study are described in . siRNA CD31 , 3′-fluorescein amidite (FAM) fluorescence-labeled siRNA CD31 (siRNA CD31 -FAM; described in ), stable negative control RNA (SNC; described in ) and RNAi-mate were all synthesized by GenePharma Co., Ltd. (Shanghai, China).

Techniques: Injection, Saline, Control, Small Interfering RNA

Journal: Oncology Letters

Article Title: Inhibition of lung tumor growth in nude mice by siRNA CD31 targeting PECAM-1

doi: 10.3892/ol.2014.2091

Figure Lengend Snippet: siRNA CD31 downregulates PECAM-1 protein expression of tumor xenografts using RNAi-mate as a carrier. The measured values (MV, μg/ml) and BCA correction values (BCV, μg/mg) of PECAM-1 in the tumor xenografts of the nude mice treated with siRNA CD31 -RNAi-mate complexes (siRNA CD31 group) decreased compared with those of the tumor xenograft of the control nude mice treated with saline (control group) (all P<0.01). The MV and BCV of PECAM-1 in other tissues (lung, liver, brain, heart and kidney) of the siRNA CD31 group were not different compared with the values of the control group (all P>0.05). Each assay was performed in triplicate and was independently repeated three times. Error bars show the standard error, n=6; * P MV <0.01 vs. control MV; # P BCV <0.01 vs. control BCV; & siRNA CD31 group, the nude mice treated with an injection of siRNA-RNAi-mate via the tail-vein; $ control group, the nude mice treated with an injection of saline via the tail-vein; siRNA, small interfering RNA; PECAM-1, platelet endothelial adhesion molecule 1; CD31, cluster of differentiation 31; RNAi, RNA interference; BCA, bicinchoninic acid, for the determination of total protein in homogenate (BCA correction value was calculated by: BCA correction value = measured value / BCA value).

Article Snippet: The 2′-O-methyl-modified siRNA CD31 molecules used in the present study are described in . siRNA CD31 , 3′-fluorescein amidite (FAM) fluorescence-labeled siRNA CD31 (siRNA CD31 -FAM; described in ), stable negative control RNA (SNC; described in ) and RNAi-mate were all synthesized by GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Control, Saline, Injection, Small Interfering RNA

Journal: Oncology Letters

Article Title: Inhibition of lung tumor growth in nude mice by siRNA CD31 targeting PECAM-1

doi: 10.3892/ol.2014.2091

Figure Lengend Snippet: siRNA CD31 weakens the VEGF protein expression of tumor xenografts using RNAi-mate as carrier. Measured values (MV, ng/ml) and BCA correction values (BCV, ng/mg) of VEGF in the tumor xenografts of the nude mice treated with siRNA CD31 -RNAi-mate complexes (siRNA CD31 group) decreased compared with those of the tumor xenografts of the control nude mice treated with saline (group control) (all P<0.01). The MV and BCV of VEGF in the other tissues (lung, liver, brain, heart and kidney) of the siRNA CD31 group were not different compared with the values of the control group (all P>0.05). Each assay was performed in triplicate and was independently repeated three times. Error bars show the standard error, n=6; * P MV <0.01 vs. control MV; # P BCV <0.01 vs. control BCV; & siRNA CD31 group, the nude mice treated with an injection of siRNA-RNAi-mate via the tail vein; $ control group, the nude mice treated with an injection of saline via the tail vein; siRNA, small interfering RNA; CD31, cluster of differentiation 31; VEGF, vascular endothelial growth factor; RNAi, RNA interference; BCA, bicinchoninic acid, for the determination of total protein in homogenate (BCA correction value was calculated by: BCA correction value = measure value / BCA value).

Article Snippet: The 2′-O-methyl-modified siRNA CD31 molecules used in the present study are described in . siRNA CD31 , 3′-fluorescein amidite (FAM) fluorescence-labeled siRNA CD31 (siRNA CD31 -FAM; described in ), stable negative control RNA (SNC; described in ) and RNAi-mate were all synthesized by GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Control, Saline, Injection, Small Interfering RNA

Journal: Journal of Translational Medicine

Article Title: An induction of microRNA, miR-7 through estrogen treatment in breast carcinoma

doi: 10.1186/1479-5876-10-S1-S2

Figure Lengend Snippet: Effects of miR-7 upon EGFR mRNA abundance in MCF-7 cell line. EGFR mRNA levels in MCF-7 transfected with miR-7 for five days. *, Significantly different from miR-NC. p < 0.05. miR-NC, transfection with scramble RNA.

Article Snippet: Scramble RNA (Genolution Pharmaceutical) was employed for negative control.

Techniques: Transfection