Journal: Genes & Cancer

Article Title: Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer

doi:

Figure Lengend Snippet: MDA-MB-468 and SUM159PT cells were transfected with siRNA targeting GRN (pool siGRN and individual constructs siGRN-B and siGRN-C) or a non-targeting control (siCon). They were then analyzed by (A) immunoblot for levels of phosphorylated and total STAT3 in whole cell lysates, (B) luciferase reporter assay for STAT3-dependent transcriptional activity (N = 3), (C) nuclear and cytoplasmic fractions (tubulin serves as a loading control for the cytoplasmic fraction and PARP serves as a loading control for the nuclear fraction), (D) qRT-PCR for expression of endogenous STAT3 target genes (normalized to GAPDH; N = 3), and (E) chromatin immunoprecipitation with an antibody for STAT3 followed by PCR at the indicated STAT3 target genes (representative of N = 3).

Article Snippet: Cells were transfected with 10 nM of siRNA targeting granulin (acrogranin; sc-39621 pool, sc-39621B 5′-GCUUCCAAAGAUCAGGUAATT-3′, sc-39621C 5′-GGACAGUACUGAAGACUCUTT-3′, Santa Cruz), STAT3 (D-003544-03-0010), or non-targeting siControl (D-001210-03-20, Dharmacon Fisher Scientific, Pittsburgh, PA), using Lipofectamine RNAiMAX (Invitrogen) for forty-eight hours.

Techniques: Transfection, Construct, Control, Western Blot, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation

Journal: Genes & Cancer

Article Title: Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer

doi:

Figure Lengend Snippet: (A) STAT3-GRN interaction in SK-BR-3 cells was analyzed by immunoprecipitation using antibodies against STAT3 (or non-specific IgG) followed by immunoblot with the indicated antibodies. Input indicates 5% of pre-immunoprecipitated samples. SK-BR-3 cells were transfected with siRNA targeting GRN (pool siGRN and individual constructs siGRN-B and siGRN-C) or a non-targeting control (siCon), then stimulated with LIF were analyzed by (B) immunoblot and (C) luciferase reporter assay for STAT3-dependent transcriptional activity. SK-BR-3 cells transfected with siRNA targeting GRN (pool) were analyzed by (D) luciferase reporter assay for STAT3-dependent transcriptional activity (B-luc, M67-luc) (N = 3), (E) qRT-PCR for expression of endogenous STAT3 target genes (normalized to HPRT; N = 6), and (F) chromatin immunoprecipitation with an antibody to STAT3 followed by PCR at the indicated STAT3 target genes (data normalized to unstimulated cells; representative of N = 2).

Article Snippet: Cells were transfected with 10 nM of siRNA targeting granulin (acrogranin; sc-39621 pool, sc-39621B 5′-GCUUCCAAAGAUCAGGUAATT-3′, sc-39621C 5′-GGACAGUACUGAAGACUCUTT-3′, Santa Cruz), STAT3 (D-003544-03-0010), or non-targeting siControl (D-001210-03-20, Dharmacon Fisher Scientific, Pittsburgh, PA), using Lipofectamine RNAiMAX (Invitrogen) for forty-eight hours.

Techniques: Immunoprecipitation, Western Blot, Transfection, Construct, Control, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation

Journal: Genes & Cancer

Article Title: Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer

doi:

Figure Lengend Snippet: (A) SK-BR-3 cells were transfected with siRNA targeting GRN or a non-targeting control, then stimulated with LIF for the indicated times. Cells were then analyzed by immunoblot for levels of phosphorylated and total STAT3 in whole cell extracts. Tubulin serves as a loading control. (B) Immunoblot band intensities plotted as ratios of phosphorylated STAT3 to tubulin. (C) Area under the curve of phospho-STAT3:tubulin (N = 5). (D) SK-BR-3 cells were transfected with siRNA targeting GRN or a non-targeting control, then stimulated with LIF for the indicated times. Cells were then analyzed by immunoblot for levels of phosphorylated and total STAT3 in nuclear fractions. PARP serves as a nuclear fraction loading control. (E) Immunoblot band intensities plotted as ratios of phospho-STAT3 to PARP (N = 4).

Article Snippet: Cells were transfected with 10 nM of siRNA targeting granulin (acrogranin; sc-39621 pool, sc-39621B 5′-GCUUCCAAAGAUCAGGUAATT-3′, sc-39621C 5′-GGACAGUACUGAAGACUCUTT-3′, Santa Cruz), STAT3 (D-003544-03-0010), or non-targeting siControl (D-001210-03-20, Dharmacon Fisher Scientific, Pittsburgh, PA), using Lipofectamine RNAiMAX (Invitrogen) for forty-eight hours.

Techniques: Transfection, Control, Western Blot

Journal: Genes & Cancer

Article Title: Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer

doi:

Figure Lengend Snippet: (A) Viable cell number was quantitated by ATP-dependent assay for MDAMB-468, SUM159PT, and SK-BR-3 cells transfected with siRNA targeting GRN (N ≥ 3). (B) Viable cell number was quantitated forSUM159PT cells transfected with siRNA targeting GRN (or control) and an expression construct for constitutively active STAT3 (S3C)or vector control (N = 2). SUM159PT cells transfected with siRNA targeting GRN (pool siGRN and individual constructs, siGRN-B andsiGRN-C) or STAT3 were analyzed by (C) clonogenicity and (D) wound healing assays.

Article Snippet: Cells were transfected with 10 nM of siRNA targeting granulin (acrogranin; sc-39621 pool, sc-39621B 5′-GCUUCCAAAGAUCAGGUAATT-3′, sc-39621C 5′-GGACAGUACUGAAGACUCUTT-3′, Santa Cruz), STAT3 (D-003544-03-0010), or non-targeting siControl (D-001210-03-20, Dharmacon Fisher Scientific, Pittsburgh, PA), using Lipofectamine RNAiMAX (Invitrogen) for forty-eight hours.

Techniques: Transfection, Control, Expressing, Construct, Plasmid Preparation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Engineering Nanoparticles for Targeted Delivery of Nucleic Acid Therapeutics in Tumor

doi: 10.1016/j.omtm.2018.09.002

Figure Lengend Snippet: The siRNA/MicroRNA-Based Drugs Targeting Different Diseases in Clinical Trials

Article Snippet: PDA, PC , siRNA target K-RAS , biodegradable polymer matrix , siG12D LODER , Silenseed , NCT01188785 (I) NCT01676259 (II).

Techniques: shRNA, Plasmid Preparation, Infection, Injection