SIHK1738

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 3×10^5 cells per well	siRNA concentratioin-50 nM PKN3 siRNA were as follows: Sense, 5′-AgA cUu GaG gAc UuC cUg GaC aA-3′ and antisense, 5′-uUg UcC aGg AaG uCc UcA aGu Cu-3′ Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).  Incubate for 5 minutes at room temperature and add to cells.  Incubate cells for 1–3 days at 37°C.

Upstream tips
Seed 3×10^5 cells per well
Protocol tips
siRNA concentratioin-50 nM PKN3 siRNA were as follows: Sense, 5′-AgA cUu GaG gAc UuC cUg GaC aA-3′ and antisense, 5′-uUg UcC aGg AaG uCc UcA aGu Cu-3′ Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).  Incubate for 5 minutes at room temperature and add to cells.  Incubate cells for 1–3 days at 37°C.

Publication protocol

PKN3 expression level in vitro
To investigate the PKN3 mRNA levels in tumor cells, total RNA was isolated from MDA-MB-231, MCF-7, PANC-1, HeLa, HepG2, LLC and Colon 26 cells using NucleoSpin RNA (Macherey-Nagel, Düren, Germany). For reverse transcription-polymerase chain reaction (RT-PCR), the 20-µl reaction volume contained 1 µl synthesized cDNA, 10 pmol of each specific primer pair (Fasmac Co., Ltd., Kanagawa, Japan) and 0.25 U Ex Taq DNA polymerase (Takara Bio, Inc., Otsu, Japan) with PCR buffer containing 1.5 mM MgCl2 and 0.25 mM of each dNTP (Takara Bio, Inc., Otsu, Japan). The profile of PCR amplification consisted of denaturation at 95°C for 0.5 min, primer annealing at 55°C for 0.5 min, and elongation at 72°C for 1 min for 30 cycles. Human PKN3 cDNA (115 bp) was amplified using the following primers: Forward, 5′-CACTTTGGGAAGGTCCTCCTG-3′ and reverse, 5′-CGCAGTACAGGCTCTCTATCT-3′. Mouse PKN3 cDNA (88 bp) was amplified using the following primers: Forward, 5′-TCACCTTCTGCGAGCCTGTC-3′ and reverse, 5′-ATGAAATCCCGGCCTCTCCGC-3′. Human GAPDH cDNA (820 bp) was amplified using the following primers: Forward, 5′-ATGACCCCTTCATTGACCTC-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAAT-3′. Mouse β-actin cDNA (514 bp) was amplified using the following primers: Forward, 5′-TGTGATGGTGGGAATGGGTCAG-3′ and reverse, 5′-TTTGATGTCACGCACGATTTCC-3′. The PCR products were analyzed by 18% acrylamide gel electrophoresis in a Tris-borate-EDTA buffer (Wako Pure Chemical Industries Inc.), and were visualized by ethidium bromide staining.

For knockdown of PKN3 mRNA by transfection of cells with PKN3 siRNA, MDA-MB-231, PANC-1, HeLa, LLC and Colon 26 cells were plated into 6-well culture dishes at a density of 3×105 cells per well. The cells were transfected with 50 nM Cont siRNA or PKN3 siRNA using Lipofectamine RNAiMax reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was isolated and quantitative RT-PCR was performed using a iCycler MyiQ detection system (Bio-Rad Laboratories, Hercules, CA, USA) and a SYBR Green I assay (iQ™ SYBER Green Supermix; Bio-Rad Laboratories) 48 h subsequent to transfection. Human and mouse PKN3 cDNAs were amplified using the aforementioned primers. Human β-actin cDNA (186 bp) was amplified using the following primers: Forward, 5′-TGGCACCCAGCACAATGAA-3′ and reverse, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. Mouse β-actin cDNA (171 bp) was amplified using the following primers: Forward, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and reverse, 5′-ATGGAGCCACCGATCCACA-3′. Samples were run in triplicate and the expression levels of human PKN3 and mouse PKN3 mRNA were normalized to the quantity of human β-actin and mouse β-actin mRNA, respectively, in the same sample, and analyzed using the comparative Cq method (16).

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