SIHK1738

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	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 cells for 48 h at 37°C.

Protocol tips
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 cells for 48 h at 37°C.

Publication protocol

siRNA Mouse/human specific PKN3 siRNA and pGL2-luciferase siRNA (Cont siRNA), as a negative control for PKN3 siRNA (blunt-ended 23-mer, alternating 2′-O-methyl modified), was synthesized by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The siRNA sequences of the 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′ (5). The siRNA sequences of the Cont siRNA were as follows: Sense, 5′-AuC aCg UaC gCg GaA uAc UuC gA-3′ and antisense, 5′-uCg AaG uAu UcC gCg UaC gUg Au-3′ (5). The lower-case letters represent 2′-O-methyl-modified nucleotides.

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).

Cell growth inhibition
MCF-7, MDA-MB-231, LLC and Colon 26 cells were seeded in 96-well plates at a density of 2×104 cells/well 24 h prior to transfection. Cells at 50% confluency were transfected with 50 nM Cont siRNA or PKN3 siRNA using Lipofectamine RNAiMax reagent and then incubated for 48 h. In the PKN3 siRNA or Cont siRNA with DXR combined treatment conditions, the cells were incubated for 2 h subsequent to transfection with 50 nM PKN3 siRNA or Cont siRNA using Lipofectamine RNAiMax reagent, and then treated with various concentrations (0.016–0.5 µM) of DXR for an additional 48 h. The cell number was determined using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cell viability was expressed relative to the absorbance of untreated cells at 450 nm.

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