Journal: BMC Biochemistry

Article Title: Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a

doi: 10.1186/s12858-015-0045-6

Figure Lengend Snippet: Kv1.3-eGFP trafficking after siRNA mediated knockdown of Sec24. Kv1.3-eGFP trafficking was examined after the knockdown of Sec24 isoforms (as indicated) in the presences of the membranous ER resident protein Sec61β tagged with the mCherry fluorophore (Sec61β-mCherry). Cellular nuclei were stained with DAPI. The wild-type (wt) trafficking profile is similar to the trafficking profile of Sec24c and Sec24cd knockdown conditions. An altered trafficking profile is seen in Sec24a, Sec24b, Sec24ab, and Sec24abcd conditions. Interestingly, there is also an altered trafficking profile in the Sec24d condition, but the Kv1.3-eGFP signal does not overlap well with the Sec61β protein. Line scans of each fluorescent channel are shown. The white arrow in the merged image represents the placement and direction of the line scan. Scale bar = 5 μm

Article Snippet: Following siRNA treatment, COS-1 cells were further co-transfected with Kv1.3-eGFP and cDNAs encoding for either the vesicular stomatitis virus glycoprotein tagged with a cerulean fluorophore (VSVG-Cerulean) (Addgene; plasmid 11913) [ ], or the membranous ER resident protein Sec61β tagged with the mCherry fluorophore (Sec61β-mCherry) (Addgene; 49155) [ ] using the LipofectamineTM 2000 reagent (Invitrogen) as described above.

Techniques: Knockdown, Staining

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 prevents cardiac dysfunction induced by pressure overload. ( A ), Echocardiographic analysis of fraction shortening (FS) in mice at different time-points (3d, 1w, 2w, and 4w) after TAC. * p < 0.05 vs Sham group; n = 6-10 mice/each group. ( B ), Western blot analysis of p-LRP6 and LRP6 levels in left ventricular tissues from mice at different time-points (3d, 1w, 2w, and 4w) after TAC. * p < 0.05; ** p < 0.01 vs sham group; n = 4 mice/ each group. ( C ), Summary of the generation of tamoxifen-inducible cardiac-specific LRP6 overexpressing mice. ( D ), Representative images of Western blot analysis of LRP6 expression in heart and kidney tissues from LRP6 CAG /MCM after injection of tamoxifen or DMSO for 3 consecutive days. ( E ), Representative images of Western blot analysis of LRP6 expression in isolated CMs and non-CMs from tamoxifen-injected LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice. CMs: cardiomyocytes. ( F ), Overall strategy of exploring the effects of cardiac LRP6 overexpression in TAC mice. ( G ), Echocardiographic analysis of cardiac function in tamoxifen-injected LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice at 2 weeks and 4 weeks after TAC or sham operation. FS: fraction shortening; EF: ejection fraction; LVID;d: left ventricular diastolic internal diameter. HR: heart rate; * p < 0.05; ** p < 0.01; n = 7-12/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Western Blot, Expressing, Injection, Isolation

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 attenuates cardiac hypertrophy and fibrosis induced by pressure overload. ( A ), HE and Masson staining of cross heart sections. Scale bars, 2 mm. ( B ), Representative images of Masson staining heart tissue were showed and the cardiac interstitial and vascular fibrosis were quantitatively analyzed. Scale bars, 100 um. ( C ), Western blot analysis of p-LRP6, LRP6, Col1, Col3, α-SMA, MMP2, MMP9, TGF-β1, p-smad2/3 in heart tissues. * p < 0.05; ** p < 0.01; *** p <0.001; n = 3-5/each group. ( D ), Quantitative analysis of the ratio of heart weight/body weight (HW/BW) and heart/tibia (HW/TL). * p < 0.05; ** p < 0.01; *** p < 0.001; n = 4-7/each group. Tamoxifen-injected-LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice were analyzed at 4 weeks after TAC or sham operation.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Staining, Western Blot, Injection

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 inhibits the expression of cardiomyocyte-derived-Wnt5a/11 under pressure overload. ( A ), Western blot analysis of LRP6 expression in CMs, and Col1, MMP2, MMP9, TGF-β1, α-SMA, and p-smad2/3 expression in CFs. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON), and the culture medium from these CMs was used to stimulate CFs. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 4/each group. ( B ), Western blot analysis of Wnt5a and Wnt11 expression in CMs and the culture medium. Stretched (MS) or control CMs were pre-transfected with LRP6 adenovirus (Ad-LRP6) or control adenovirus (Ad-CON). ** p < 0.01; *** p < 0.001; n = 5-6/each group. ( C ), Western blot analysis of Wnt5a and Wnt11 expression in tamoxifen-injected-LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice after TAC or sham operation. ** p < 0.01; n = 5/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Expressing, Derivative Assay, Western Blot, Control, Transfection, Injection

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Knockdown of Wnt5a and Wnt11 inhibits cardiac fibrosis in mice under pressure overload. ( A ), Overall strategy to explore cardiac remodeling after knockdown of Wnt5a and Wnt11 in mice under pressure overload. ( B ), Fraction shortening (FS) in mice at 4 weeks after TAC. Mice were injected with sh-Wnt5a/Wnt11-AAV9 (sh-Wnt5a/Wnt11) or sh-scramble-AAV9 (shNC) by tail vein two weeks before TAC or sham operation. * p < 0.05; n = 6-10/each group. ( C ), Masson staining of heart tissue from mice in (B) and fibrosis area was quantitatively analyzed. Bar = 100 µm. * p < 0.05; *** p < 0.001. n = 4-9/each group. ( D ), Western blot analysis of Wnt5a, Wnt11, Col1, Col3, MMP9, TGF-β1, α-SMA, and p-smad2/3 expression in heart tissue from TAC or sham mice pre-injected with sh-Wnt5a/Wnt11-AAV9 (sh-Wnt5a/Wnt11) or sh-scramble-AAV9 (sh-NC) by tail vein. ** p < 0.01; *** p < 0.001. n = 4-6 mice/each group. (E), Western blot analysis of Col1, MMP2, MMP9, TGF-β1 and α-SMA expression in CFs stimulated with the culture medium from control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6), Wnt5a, Wnt11, Wnt5a+Wnt11 at 10 ng/mL, or PBS were supplemented with the culture medium from Ad-LRP6-CMs under MS and treated with CFs. * p < 0.05; ** p < 0.01; *** p < 0.001. n = 3/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Knockdown, Injection, Staining, Western Blot, Expressing, Control

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Cardiomyocyte-specific overexpression of LRP6 promotes the degradation of Wnt5a and Wnt11 by interaction with LRP6 and CTSD during pressure overload. ( A ), Western blot analysis of LRP6 and CTSD expression in tamoxifen-injected LRP6 CAG /MCM (LRP6 Over) or -MCM (MCM) mice at 4 weeks after TAC or sham operation. * p < 0.05; ** p < 0.01; n = 4 mice/each group. ( B ), Western blot analysis of LRP6 and CTSD expression in control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6) with or without MS. *** p < 0.001; n = 4-6/each group. ( C ), Interaction of LRP6 and CTSD was analyzed by IP analysis in control (Ad-CON) or LRP6-overexpressing CMs (Ad-LRP6) with or without MS. M: marker. ( D ), Western blot analysis of Wnt5a, Wnt11, and CTSD expression in stretched control (Ad-CON) or LRP6 overexpressing CMs (Ad-LRP6) after treatment with si-CTSD or si-scramble. * p < 0.05; *** p < 0.001; n = 3/each group. ( E ), Western blot analysis of Wnt5a and Wnt11 expression in stretched control (Ad-CON) or LRP6-overexpressing CMs (Ad-LRP6) after treatment with PBS or leupeptin (50 µg/ml). * p < 0.05; ** p < 0.01; *** p < 0.001; n = 3 /each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Western Blot, Expressing, Injection, Control, Marker

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Protease inhibitor attenuates the interaction of LRP6 and CTSD in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. ( A ), Schematic of the experimental procedure in (B). A protease inhibitor, leupeptin (40 mg/kg), was intraperitoneally injected into LRP6 Over or -MCM mice from 2 to 4 weeks (every other day) after TAC; PBS treatment was used as a control. ( B ), IP analysis of the interaction among LRP6, CTSD, Wnt5a, and Wnt11. The experiment was repeated for at least 3 times.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Protease Inhibitor, Injection, Control

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Protease inhibitor attenuates cardiac protection in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. ( A ), Western blot analysis of p-LRP6, LRP6, Wnt5a, Wnt11, Col1, Col3, MMP9 and TGF-β1 in the heart tissue. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 3/each group. ( B ), Masson staining of heart tissue and quantitative analysis of fibrosis area. The upper lane: bar = 2 mm; The lower lane: bar = 100 µm, * p < 0.05; ** p < 0.01; n = 7-10 /each group. ( C ), Fraction shortening (FS) in mice. * p < 0.05; ** p < 0.01; n = 8/ each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Protease Inhibitor, Western Blot, Staining

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Mutants of LRP6 or CTSD inhibit the interaction of LRP6 and CTSD. ( A ), Sequencing results of LRP6 and CTSD mutations. ( B ), Schematic diagram of LRP6 and CTSD showing approximate locations of the mutants. ( C ), IP analysis of the interaction among LRP6 WT, LRP6 (P1104S), or LRP6 (P1427Q) and CTSD WT or CTSD (G136R). LRP6 WT and CTSD WT or their mutant plasmids were transfected into 293T cells for 48 h. The experiment was repeated 4 times. ( D ), Western blot analysis of LRP6, CTSD, Wnt5a, Wnt11, and MESD in stretched COS7 cells transfected with LRP6 WT and CTSD WT or their mutant plasmids as in (C). **** P < 0.0001, n = 3/each group.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Sequencing, Mutagenesis, Transfection, Western Blot

Journal: Theranostics

Article Title: Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis

doi: 10.7150/thno.48787

Figure Lengend Snippet: Proposed model of cardiac protection mediated by cardiac-specific LRP6 overexpression in response to pressure overload. LRP6 overexpression suppresses β-catenin activation, which may contribute to the inhibition of cardiac hypertrophy. LRP6 overexpression inhibits cardiomyocyte-derived Wnt5a/Wnt11 to inhibit cardiac fibrosis. Cardiomyocyte-expressed LRP6 interacts with CTSD, which may promote the degradation of Wnt5a and Wnt11.

Article Snippet: Human wild type LRP6 was obtained from Addgene (LRP6-pCS2-VSVG, 27282#).

Techniques: Over Expression, Activation Assay, Inhibition, Derivative Assay