polyjet tm reagent sl100688  (SignaGen)

Journal: The EMBO Journal

Article Title: ArfGAP1 inhibits mTORC1 lysosomal localization and activation

doi: 10.15252/embj.2020106412

Figure Lengend Snippet: Wild‐type (WT) human embryonic kidney 293A (HEK293A) cells transfected with HA‐tagged ArfGAP1 or RFP1 (control) were plated, and cell numbers were counted at 48 and 96 h. Three replicated wells were counted each group. Columns represent Mean ± SEM. Student’s t ‐test P = 0.021 at 48 h and P = 0.058 at 96 h. WT HEK293A cells were transfected with HA‐tagged ArfGAP1 or HA‐tagged RFP1 (control) for 48 h, and cell diameters were determined using a particle size counter. The curves show the overall distribution of cell size in each group. Mean ± SD ( N = 4): RFP1 17.64 ± 0.03 and ArfGAP1: 17.48 ± 0.12. Student’s t ‐test P = 0.046. Left— Mouse PaCa cells stably overexpressing HA‐tagged ArfGAP1 or HA‐tagged RFP1 (control) were plated and cell numbers counted on Day 1, 2, and 3. Six replicated wells were counted each group. Bars represent Mean ± SEM. Student’s t ‐test P = 0.005 on Day 3. Right— Overexpression of HA‐tagged ArfGAP1 or HA‐tagged RFP1 (control) was confirmed by immunoblotting of HA. ArfGAP1 antibody shows both endogenous (lower band) and overexpressed (upper band) ArfGAP1. Vinculin is a loading control. Left— MIA Paca‐2 cells were transfected with small inference RNAs (siRNAs) against ArfGAP1 or a negative control siRNA (siControl) twice (24 and 72 h after cell seeding). On the following day of second transfection (Day 0), cells were plated and cell numbers were counted at Day 1, 2, and 3. Six replicated wells were counted each group. Bars represent Mean ± SEM. Student’s t ‐test P = 0.009 for siControl versus siArfGAP1‐1 and P = 0.003 for siControl versus siArfGAP1‐3 at Day 3. Right— MIA Paca‐2 cells were transfected with siRNAs as above described, and ArfGAP1 protein levels were confirmed by immunoblotting. Vinculin serves as a loading control.

Article Snippet: Cells were transfected with plasmid DNA using PolyJet TM DNA In Vitro Transfection Reagent (#SL100688 from SignaGen Laboratories) according to manufacturer’s instructions.

Techniques: Transfection, Control, Stable Transfection, Over Expression, Western Blot, Negative Control

Journal: Oncogene

Article Title: Overexpressed miR-200a promotes bladder cancer invasion through direct regulating Dicer/miR-16/JNK2/MMP-2 axis

doi: 10.1038/s41388-019-1120-z

Figure Lengend Snippet: miR-200a overexpression resulted in C-Jun phosphorylation, and its regulated MMP-2 transcription and protein expression. a , b Total RNA was extracted from cells and MMP-2 mRNA levels evaluated using real-time PCR. Wild-type MMP-2 promoter-driven luciferase reporter was co-transfected together with pRL-TK into T24T(Vector) and T24T(miR-200a) cells ( c ), or UMUC3(Vector) and UMUC3(miR-200a) cells ( d ). After 24 h of transfection, luciferase activity was evaluated. TK was used as an internal control. Results presented as MMP-2 promoter activity relative to control vector transfectant. Each bar represents means ± SD of three independent experiments. e Potential transcription factor binding sites in human MMP-2 promoter region. Extracts obtained from T24T(Vector) vs. T24T(miR-200a) ( f ) or UMUC3(Vector) vs. UMUC3(miR-200a) ( g ) cells were analyzed for activation/expression of transcription factors indicated. h TAM67 and vector control were stably transfected into T24T(miR-200a) cells and transfectants were identified by western blot. i TAM67 and its vector control were stably transfected into T24T(miR-200a) cells; total RNA was extracted, and MMP-2 mRNA levels were evaluated using real-time PCR). Bars represent means ± SD from three independent experiments. j Wild-type MMP-2 promoter-driven luciferase reporter was co-transfected together with pRL-TK into T24T(miR-200a/Vector) and T24T(miR-200a/TAM67) cells. After 24 h of transfection, cells were extracted for determination of luciferase activity. Results were presented as MMP-2 promoter activity relative to vector control transfectants. Each bar represents means ± SD from three independent experiments. * p < 0.01

Article Snippet: Cell transfections were performed with PolyJet TM DNA in vitro Transfection Reagent (# SL100688, SignaGen Laboratories, Rockville, MD, USA).

Techniques: Over Expression, Phospho-proteomics, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Control, Binding Assay, Activation Assay, Stable Transfection, Western Blot

Journal: Oncogene

Article Title: Overexpressed miR-200a promotes bladder cancer invasion through direct regulating Dicer/miR-16/JNK2/MMP-2 axis

doi: 10.1038/s41388-019-1120-z

Figure Lengend Snippet: miR-200a overexpression specifically resulted in JNK2 induction, in turn leading to c-Jun phosphorylation and MMP-2 transcription. a – d Cell extracts obtained from the indicated transfectants were subjected to western blot for determination of protein expression. β-Actin or α-tubulin were used as protein loading control. e shJNK2 and Nonsense were stably transfected into T24T(miR-200a) cells and total RNA was extracted; MMP-2 mRNA levels in cells were evaluated using real-time PCR. Bars represent means ± SD from three independent experiments. f Wild-type MMP-2 promoter-driven luciferase reporter was co-transfected together with pRL-TK into T24T(miR-200a/nonsense) and T24T(miR-200a/shJNK2) cells. After 24 h of transfection, transfectants were used to evaluate for luciferase activity. Results presented as MMP-2 promoter activity relative to vector control transfectants. Each bar represents means ± SD from three independent experiments. Invasion abilities of T24T(miR-200a/Nonsense) vs. T24T(miR-200a/shJNK2) cells and UMUC3 (miR-200a/Nonsense) vs. UMUC3 (miR-200a/shJNK2) cells were evaluated using invasion chamber ( g , i ); relative invasion ability was plotted ( h , j ). Bars represent means ± SD from three independent experiments. *Significant difference ( p < 0.01)

Article Snippet: Cell transfections were performed with PolyJet TM DNA in vitro Transfection Reagent (# SL100688, SignaGen Laboratories, Rockville, MD, USA).

Techniques: Over Expression, Phospho-proteomics, Western Blot, Expressing, Control, Stable Transfection, Transfection, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Plasmid Preparation

Journal: Oncogene

Article Title: Overexpressed miR-200a promotes bladder cancer invasion through direct regulating Dicer/miR-16/JNK2/MMP-2 axis

doi: 10.1038/s41388-019-1120-z

Figure Lengend Snippet: miR-200a inhibited miR-16 expression, therefore reducing miR-16 binding to 3′-UTR of JNK2 mRNA, and increasing JNK2 protein translation. a JNK2 3′-UTR activity was evaluated by transfection of JNK2 3′-UTR-driven luciferase reporter along with pRL-TK into transfectants as indicated. Bars represent means ± SD from three independent experiments. b miRNA expression levels were evaluated by real-time PCR in T24T(Vector) vs. T24T(miR-200a) cells. Results were normalized to U6. c miRNA expression levels were evaluated by real-time PCR in T24T(Vector) vs. T24T (miR-200a inhibitor) cells. Results were normalized to U6. d Schematic of miR-16 binding site in JNK2 mRNA 3′-UTR region and its mutants aligned with miR-16. e T24T(Vector) and T24T(miR-200a) cells were co-transfected with wild-type and mutant JNK2 3′-UTR luciferase reporters and pRL-TK, respectively. Luciferase activity of each transfectant was evaluated and results were presented as relative JNK2 3′-UTR activity. f Real-time PCR was used to identify the miR-16 expression in T24T(miR-200a/Vector) vs. T24T (miR-200a/ miR-16) cells. Bars represents means ± SD from three independent experiments. g Extracts from T24T (miR-200a/ Vector) vs. T24T (miR-200a/ miR-16) cells were used to evaluate effect of miR-16 on expression of JNK1, JNK2, p-c-Jun, c-Jun, and MMP-2 by western blot. β-Actin was used as protein loading control. Results shown are representative of three independent experiments. Invasion abilities of T24T(miR-200a/Vector) and T24T(miR-200a/miR-16) cell were evaluated using Invasion Chambers ( h ); and the relative invasion ability was plotted ( i ). Bars represent means ± SD from three independent experiments. j Real-time PCR was used to identify the miR-16 expression in T24T(Vector) vs. T24T (miR-16 inhibitor) cells. Bars represents means ± SD from three independent experiments. k Extracts from T24T(Vector) vs. T24T (miR-16 inhibitor) cells were used to evaluate effect of miR-16 on expression of JNK1, JNK2, p-c-Jun, c-Jun, and MMP-2 by western blot. β-Actin was used as protein loading control. Results shown are representative of three independent experiments. Invasion abilities of T24T(Vector) and T24T (miR-16 inhibitor) cell were evaluated using invasion chambers ( l ); and the relative invasion ability was plotted ( m ). Bars represent means ± SD from three independent experiments. * p < 0.01, # p < 0.05

Article Snippet: Cell transfections were performed with PolyJet TM DNA in vitro Transfection Reagent (# SL100688, SignaGen Laboratories, Rockville, MD, USA).

Techniques: Expressing, Binding Assay, Activity Assay, Transfection, Luciferase, Real-time Polymerase Chain Reaction, Plasmid Preparation, Mutagenesis, Western Blot, Control