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1.
FIGURE 2.

FIGURE 2. From: Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel ?1C Subunit in Chicken Cone Photoreceptors.

Overexpression of gga-mir-26a reduced L-VGCCα1C expression in chicken cone photoreceptors. Chicken cone photoreceptors were co-transfected with a plasmid encoding eGFP and empty vector (pSilencer; A) (negative control), gga-mir-26a (pSilencer-mir-26a) (B), or gga-mir-124a (pSilencer-mir-124a; C) (positive control). I, phase-contrast images of cone photoreceptors; the white arrowheads indicate the oil droplets, and the black arrowheads indicate the gold particles used for biolistic transfection. The white arrows indicate the cone photoreceptors that were positively transfected. II, expression of eGFP in transfected photoreceptors (white arrows). III, immunocytochemical staining of L-VGCCα1C; the white arrows indicate photoreceptors that were positively transfected. IV, overlay images of both eGFP- and L-VGCCα1C-positive. D, the fluorescence intensity of L-VGCCα1C was quantified. There was no difference in L-VGCCα1C fluorescence intensity between control (non-transfected) photoreceptors, photoreceptors transfected with empty vector, or photoreceptors transfected with gga-mir-124a. In gga-mir-26a-transfected cone photoreceptors, there is a significant decrease in L-VGCCα1C fluorescence intensity, whereas transfection with gga-mir-124a has no effect on the expression of L-VGCCα1C. Each group contained four trials. *, p < 0.05.

Liheng Shi, et al. J Biol Chem. 2009 September 18;284(38):25791-25803.
2.
FIGURE 1.

FIGURE 1. From: Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel ?1C Subunit in Chicken Cone Photoreceptors.

gga-mir-26a targeted the 3′-UTR region of L-VGCCα1C mRNA. A, the L-VGCCα1C 3′-UTR is highly conserved among humans, mice, and chickens. The arrow indicates the mir-26a target site. B, the naive (wild type) L-VGCCα1C 3′-UTR sequence containing the mir-26a target site was amplified, and four repeats were constructed into a pTK-Luc reporter vector (TK-Luc4XWT). The multimers with a selected point mutation in the L-VGCCα1C 3′-UTR mir-26a target sequence (TK-Luc4Xmt) was also constructed as negative controls for the same reporter assays. Luciferase reporter assays were performed after co-transfection with TK-Luc4XWT (left) or TK-Luc4Xmt (right) along with an empty vector (pSilencer; black bars), gga-mir-26a (pSilencer-gga-mir-26a; light gray bars), or gga-mir-124a (pSilencer-gga-mir-124a, positive control; diagonal lined bars). gga-mir-26a significantly inhibited the naive TK-Luc4XWT luciferase activity compared with the empty vector or gga-mir-124a. gga-mir-26a did not affect mutant TK-Luc4Xmt luciferase activity. n = 5 for each group. *, p < 0.05. RLU, relative luciferase units.

Liheng Shi, et al. J Biol Chem. 2009 September 18;284(38):25791-25803.
3.
FIGURE 5.

FIGURE 5. From: Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel ?1C Subunit in Chicken Cone Photoreceptors.

There was a diurnal expression of gga-mir-26a in the chicken retina. A, the top shows the sequence alignment of mature mir-26a among human (Hsa), mouse (Mmu), dog (Rno), chicken (Gga), and Drosophila (Dre). *, identical nucleotides. The lower panel shows the RT-PCR amplification of gga-mir-26a precursor from the chicken retina harvested at ZT 5 (day) and ZT 17 (night). β-Actin served as an internal control. B, there were diurnal expressions of mature gga-mir-26a (mir-26a) and mRNAs of Bmal1 and L-VGCCα1C in the chicken retina. Upper left, gga-mir-26a reached its highest levels at ZT 4, which was significantly different from the other five time points (n = 5 for each time point; *, p < 0.05). Upper right, the mRNA levels of Bmal1 at ZT 8 and 12 are significantly different from ZT 0, 4, 16, and 20 (n = 6 for each time point; *, p < 0.05). Lower left, the mRNA levels of L-VGCCα1C at ZT 12 and 16 are significantly higher than ZT 0, 4, 8, and 20 (n = 6 for each time point; *, p < 0.05).

Liheng Shi, et al. J Biol Chem. 2009 September 18;284(38):25791-25803.
4.
FIGURE 3.

FIGURE 3. From: Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel ?1C Subunit in Chicken Cone Photoreceptors.

gga-mir-26a repressed L-VGCC current amplitudes. Photoreceptors (A, B, and E) or cardiomyocytes (C, D, and F) were co-transfected with eGFP and either the empty vector (control) or gga-mir-26a. A, B, and E, after 24 h in DD, perforated patch recordings were performed on photoreceptors only at night (ZT 16–19). Two representative recordings from photoreceptors transfected with empty vector (control) or gga-mir-26a (A and B). A, cells were held at −65 mV, and step commands were given from −60 to +50 mV at 10-mV intervals in 50 ms. B, a ramp command was given from −80 to +60 mV over 500 ms. E, the average current density (pA/pF)-voltage (mV) relationship obtained from step commands of control (solid circle) or gga-mir-26a (solid square) transfected photoreceptors were plotted. n = 9 for the control, and n = 8 for mir-26a; *, p < 0.05 in Student's t test. Whole cell patch recordings from two representative cardiomyocytes were transfected with empty vector (control) or gga-mir-26a (C and D). C, cells were held at −65 mV, and step commands were given from −60 to +50 mV at 10-mV intervals in 200 ms. D, a ramp command was given from −80 to +60 mV over 500 ms. F, the average current density (pA/pF)-voltage (mV) relationship of control (open circle) or mir-26a (open square) transfected cardiomyocytes was plotted. n = 8 for both control and mir-26a groups; *, p < 0.05 in Student's t test.

Liheng Shi, et al. J Biol Chem. 2009 September 18;284(38):25791-25803.
5.
FIGURE 6.

FIGURE 6. From: Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel ?1C Subunit in Chicken Cone Photoreceptors.

CLOCK and CREB enhance gga-mir-26a expression in vitro. The luciferase reporter assays were performed by co-transfecting COS1 cells with one of the four reporter constructs (I, II, III, or IV) with either empty (pCDNA3.1), CLOCK (pSG5-CLOCK), or CREB (pCMV-CREB) expression vector. A, the sequence upstream of the gga-mir-26a promoter region (−1120 to −173) contained two CRE (italic type) and two E-box (italic and underlined type) cis elements. B, schematic diagram of the four luciferase reporter constructs of the gga-mir-26a promoter region: luciferase reporter with the 2-kb gga-mir-26a promoter region (I); 2-kb promoter region with a deletion mutation of E-box binding sites (Δ−330 to −150) (II); shorter promoter region with CRE and E-box deletion mutations (−150 to +1) (III); another E-box deletion mutation within the promoter region from −2 kb to −300 (IV). C, relative luminescence intensity (RLU) from the luciferase reporter assay after co-transfection with one of the four reporter constructs (I, II, III, or IV) and empty vector pCDNA3.1 (dark gray), pSG5-CLOCK (light gray), or pCMV-CREB (diagonal lines). Luciferase reporter assay results showed that CLOCK enhanced gga-mir-26a expression 3-fold, whereas CREB increased it 2-fold. Deletion of E-box (II, III, and IV) or both E-box and CRE (III) elements in the gga-mir-26a promoter region significantly reduced the transcriptional activities by CLOCK and CREB transcriptional factors. The empty vector pCDNA3.1 served as a control. n = 5 for each group. *, significant differences in RLU between pSG5-CLOCK or pCMV-CREB and pCDNA3.1 when co-transfected with full-length gga-mir-26a (I). #, significant decreases in RLU when pSG5-CLOCK is co-transfected with II, III, or IV compared with co-transfection with I. &, significant decrease in RLU when pCMV-CREB is co-transfected with III compared with co-transfection with I. p < 0.05.

Liheng Shi, et al. J Biol Chem. 2009 September 18;284(38):25791-25803.
6.
FIGURE 4.

FIGURE 4. From: Rhythmic Expression of MicroRNA-26a Regulates the L-type Voltage-gated Calcium Channel ?1C Subunit in Chicken Cone Photoreceptors.

Anti-gga-mir-26a increased L-VGCC current amplitudes. Photoreceptors were co-transfected with eGFP and either the empty vector (control) or an antagomir specifically against gga-mir-26a (anti-mir-26a). After 24 h in DD, perforated patch recordings were performed on photoreceptors only during the day (ZT 4–8). Shown are two representative recordings from photoreceptors transfected with empty vector (control) or anti-mir-26a (A and B). A, cells were held at −65 mV, and step commands were given from −60 to +50 mV at 10-mV intervals in 50 ms. B, a ramp command was given from −80 to +60 mV over 500 ms. C, the L-VGCC current densities recorded from naive photoreceptors (Control) or photoreceptors transfected with anti-mir-26a were blocked by extracellular administration of an L-VGCC inhibitor, nitrendipine (3 μm). D, the average current density (pA/pF)-voltage (mV) relationship obtained from step commands of control (solid circle) or anti-mir-26a (open square) transfected photoreceptors was plotted. n = 11 for the control, and n = 12 for anti-mir-26a; *, p < 0.05 in Student's t test. E, the maximal current densities of L-VGCCs were compared among four different groups: control photoreceptors recorded during the day (ZT 4–8), control photoreceptors recorded at night (ZT 16–19), anti-mir-26a-transfected photoreceptors recorded during the day (ZT 4–8), and mir-26a-transfected photoreceptors recorded at night (ZT 16–19). There is a diurnal regulation of L-VGCCs, and the maximal current density recorded during the day (ZT 4–8) is significantly different from the photoreceptors recorded at night (ZT 16–19). Recordings from cells transfected with anti-mir-26a during the day (ZT 4–8) have a significant increase in maximal L-VGCC current density compared with the day controls. Recordings from cells transfected with mir-26a at night (ZT 16–19) have a significant decrease in maximal L-VGCC current density compared with the night controls. The ZT 16–19 control and mir-26a data are the same from Fig. 3E. Comparisons were made by one-way ANOVA with Tukey's post hoc test; *, p < 0.05.

Liheng Shi, et al. J Biol Chem. 2009 September 18;284(38):25791-25803.

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