Supplemental Materials
Li et al. Protein kinase D3 is a pivotal activator of pathological cardiac hypertrophy by selectively upregulating the expression of hypertrophic transcription factors.
Table S1. Sequences of small interfering RNAs used in this study
Genes / Sense (5'-3') / Antisense (5'-3')PKD1 / GGAGGGCGAUCUUAUUGAATT / UUCAAUAAGAUCGCCCUCCTG
CAGCAAACGUAGUGUAUUATT / UAAUACACUACGUUUGCUGTA
PKD2 / CCUUCCUUAUACAUAGCUATT / UAGCUAUGUAUAAGGAAGGTG
CGGGCUGAAUUACCACAAATT / UUUGUGGUAAUUCAGCCCGCA
PKD3 / GGAUGUGGCUAUUAAAGUATT / UACUUUAAUAGCCACAUCCCT
GAUGGAUAUUGACAGUAAUTT / AUUACUGUCAAUAUCCAUCGG
CaN / GGCGAUUGAUCCCAAGUUGTT / CAACUUGGGAUCAAUCGCCTT
AGCGCUACUGUUGAGGCCATT / UGGCCUCAACAGUAGCGCUTT
NFATc1 / CCUGCAACAAGCGCAAAUATT / UAUUUGCGCUUGUUGCAGGTT
GUCCCUAUCAAGUCUCGAATT / UUCGAGACUUGAUAGGGACTT
NFATc2 / GGAAGCUACAGUGGAUAAATT / UUUAUCCACUGUAGCUUCCTT
CCACUAGCUAUGAGAAGAUTT / AUCUUCUCAUAGCUAGUGGTT
NFATc3 / CCGCCUCCAUCUACUUUAATT / UUAAAGUAGAUGGAGGCGGTT
GAGGCCACGAAAUGAUUGUTT / ACAAUCAUUUCGUGGCCUCTT
NFATc4 / GGAGUCUGAACUUAAUGAATT / UUCAUUAAGUUCAGACUCCTT
GCUGUAGUCAGUGGUACUATT / UAGUACCACUGACUACAGCTT
NC / UUCUCCGAACGUGUCACGUTT / ACGUGACACGUUCGGAGAATT
Table S2. Primer sequences used for RT-PCR and real-time PCR
Genes / Forward primer (5'-3') / Reverse primer (5'-3') / SizePKD1* / TGGCATCAGCAGACCCTTTC / TGCATTCCAGCTCGCGTAA / 420bp
PKD2* / ATCACCGCCAATGTCACCTACT / GTTTCTCCATCACCACGAATACC / 481bp
PKD3* / ATGGCAGTAGGGGCTTGGAT / GCAGCGAGAACAGGGTTATGAG / 431bp
GADPH* / CAAGTTCAACGGCACAGTCAAG / CACAGTCTTCTGAGTGGCAGTGAT / 403bp
PKD1 / GATGTTGTCATGGAAGAAGGGAG / GCGGTATGTTGTTGCTCGTAGA / 194bp
PKD2 / ATCACCGCCAATGTCACCTACT / CCATAGACCACTCCAAACTGTCC / 146bp
PKD3 / CATAACCCTGTTCTCGCTGC / TCTTTGTGGTCCTTCCCCTG / 139bp
NFATc4 / TACAGCAACAAGCGGGTGTC / CGGAGAGATGAGTCTGGTAGG / 139bp
GATA4 / TCTTGACTGAGTTCTGGGCATC / GCAACAATCTTTAGGCTCTGGT / 137bp
Nkx2.5 / CCCAACCGCCCCTACATT / CGTCTGTCTCGGCTTTGTCC / 127bp
MEF2D / GAGCAGAGCCCCCTGCTGGAGGA / TAGCAGGCCGCTGGGGCAGGCCC / 279bp
18S rRNA / ACCGCAGCTAGGAATAATGGA / GCCTCAGTTCCGAAAACCA / 163bp
ANF / GGGGGTAGGATTGACAGGAT / CTCCAGGAGGGTATTCACCA / 172bp
bMHC / CCTCGCAATATCAAGGGAAA / TACAGGTGCATCAGCTCCAG / 197bp
* Primers used for RT-PCR.
Table S3. Antibodies used in this study
Antibodies / Host / Company / Catalog No.Anti-PKD1 / Rabbit / Santa cruz / SC-935
Anti-PKD2 / Rabbit / Bioworld / BS1584
Anti-PKD3 / Goat / Santa cruz / SC-33407
Anti-CaMK2d / Goat / Santa cruz / SC-5392
Anti-ANF / Mouse / Santa cruz / SC-80686
Anti-HDAC5 / Rabbit / Cell signaling / 2082
Anti-GADPH / Mouse / Santa cruz / SC-47724
Anti-NFATc4 / Rabbit / Santa cruz / SC-13036
Anti-a-actinin / Mouse / Sigma-Aldrich / A7811
Anti-histone H3 / Rabbit / Bioworld / BS1750
Alexa fluor® 488 / Goat / Invitrogen / A11055
Alexa fluor® 488 / Rabbit / Invitrogen / A11034
Alexa fluor® 488 / Mouse / Invitrogen / A21202
Alexa fluor® 546 / Rabbit / Invitrogen / A10040
Table S4. Determination of siRNA specificity
Fold change** / NC / siPKD1 / siPKD2 / siPKD3PKD1 / 1.01(0.08) / 0.23(0.04)* / 1.03(0.08) / 0.94(0.11)
PKD2 / 1.00(0.07) / 1.10(0.12) / 0.17(0.05)* / 0.97(0.06)
PKD3 / – / – / – / –
* n = 3, p < 0.01 vs NC, ANOVA. Values in parenthesis are SEM.
PKD3 mRNA was not reliably detected.
** qPCR was used to measure mRNA.
Figure S1. Determination of PKD antibody specificity.
The three PKD antibodies chosen in the study are anti-PKD1 from Santa Cruz (SC-935), anti-PKD2 from Bioworld (BS1584), and anti-PKD3 from Santa Cruz (SC-33407). A. Detection of endogenous and over-expressed PKD isoforms in HEK293 cells using Western blot assays. These antibodies recognize endogenous antigens with apparent molecular weights of ~ 100, ~ 100 and ~90 kD. The projected molecular weights of PKD1, PKD2 and PKD3 are 97, 96 and 90 kD, respectively. The band densities of endogenous proteins were enhanced only for cells over-expressing PKD1 probed with anti-PKD1, PKD2 with anti-PKD2, and PKD3 with anti-PKD3. Note PKD1 and PKD2 present as double bands in 10% PAGE. OE, over-expression. MW, molecular weight marker. B. Immunofluorescent detection of PKD isoforms over-expressed in HEK293 cells. Fluorescent signals for endogenous PKDs were adjusted to an invisible level. Note anti-PKD1 and anti-PKD3 detected signals both in the nucleus and cytoplasm while anti-PKD2 mainly in cytoplasm and plasma membrane. C. Gene silencing of PKD1 or PKD2 in neonatal cardiomyocytes resulted in specific attenuation in fluorescent signals probed with anti-PKD1 or anti-PKD2. Silencing of PKD3 did not obviously affect fluorescent density conferred by anti-PKD1 or anti-PKD2.
Figure S2. Inhibition of PKA blunted isoproterenol-induced expression upregulation of ANF and bMHC. mRNAs of ANF (A) and bMHC (B) were induced by ISO for 48 h and measured with qPCR. H89 (2 mM) or Rp-8-Br-cAMP (10 mM) were added 30 min before ISO treatment, and remained in the culture throughout the experiment. The mean value for expression of each gene in NS-treated groups is defined as 1 (n = 3, * p < 0.01 vs NS, ANOVA). NS, normal saline.
Figure S3. Determination of siRNA transfection efficiency in neonatal cardiomyocytes.
Fluorescent FAM-conjugated negative control siRNA (green) was used to estimate the transfection efficiency and Lipofectamine 2000 was used as transient transfection medium. Propidium iodine (PI) was used as counterstain for nuclei (red). Scale bar = 25 mm.
Figure S4. Silencing PKD2 failed to blunt isoproterenol-induced expression upregulation of ANF (A) and bMHC (B).
mRNAs of ANF (A) and bMHC (B) were induced by ISO for 48 h and measured with qPCR. The mean value for expression of each gene in NC groups is defined as 1 (n = 3, * p < 0.01 vs NC, ANOVA). NC, negative control siRNA sequence.
Figure S5. Determination of siRNA transfection efficiency in neonatal cardiomyocytes.
pcDNA3.1-EGFP was used to estimate the t ransfection efficiency and transfection was achieved using the electroporation nucleofector kit (Amaxa Biosystems). Scale bar = 25 mm.
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