Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β Regulates Basal Transcriptional Regulatory Machinery to Control Cell Proliferation and Differentiation in Cranial Neural Crest-derived Osteoprogenitor Cells

doi: 10.1074/jbc.m109.035105

Figure Lengend Snippet: FIGURE 2. Loss of Tgfbr2 in CNCC results in decreased type D cyclin-dependent cell proliferation during intramembranous ossification. A, ratio of bromodeoxyuridine (BrdU)-labeled nuclei in the maxilla of Tgfbr2fl/fl (white bars) and Tgfbr2fl/fl;Wnt1-Cre (black bars) mice at E13.5 and E14.5. Data are mean S.D. values of five mice in each group. ***, p 0.001. B, immunoblotting analysis of Tgfbr2fl/fl and Tgfbr2fl/fl;Wnt1-Cre maxilla at E12.5, E13.5, and E14.5. Data shown are representative of three separate experiments. C, plot shows the ratios between cyclin D1, cyclin D2, cyclin D3, cyclin E, and cyclin A versus GAPDH based on quantitative densitometry of immunoblotting data in B; *, p 0.05. Tgfbr2fl/fl, white bars; Tgfbr2fl/fl;Wnt1-Cre, black bars. D and E, quantitative RT-PCR analyses of cyclin D regulators from E13.5 maxilla of Tgfbr2fl/fl (open columns) and Tgfbr2fl/fl;Wnt1-Cre (closed columns) mice. *, p 0.05. F, immunoblotting analysis of FoxO family members and activated JNK in the maxilla of Tgfbr2fl/fl and Tgfbr2fl/fl;Wnt1-Cre maxilla at E12.5, E13.5, and E14.5. Data shown are representative of three separate experiments. G, plot shows the ratios between FoxO3a, FoxO4, JNK, and phosphorylated JNK versus GAPDH after quantitative densitometry of immunoblotting data in F; *, p 0.05. Tgfbr2fl/fl, white bars; Tgfbr2fl/fl;Wnt1-Cre, black bars.

Article Snippet: Antibodies used for Western blotting were as follows: rabbit polyclonal antibodies against cyclin D1, cyclin D2, cyclin D3, cyclin A, cyclin E, JNK, and phospho-JNK (Cell Signaling Technology); FoxO4, FoxO3a, and Taf1 (Abcam); osteopontin, osteocalcin, and osteonectin (Santa Cruz Biotechnology); and mouse monoclonal antibody against GAPDH (Chemicon).

Techniques: Labeling, Western Blot, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Cyclin D1/D2–CDK4 Drives Cell Migration by Orchestrating Cytoskeletal Dynamics Through a TGFβ–FAK–Rac1 Axis

doi: 10.3390/ijms27031228

Figure Lengend Snippet: Co-localization of CDK4 with cyclin D1/D2 at the lamellipodia region. ( A ) Subcellular localization of CDK4 and CDK6 in wild-type HeLa cells. Cells were stained for F-actin (FITC-phalloidin, green), CDK4 or CDK6 (purple), and nuclei (DAPI, blue). Scale bar, 10 μm. ( B ) The line-scan profile shows the fluorescence intensity across the membrane ruffles, cytoplasm, and nucleus along the line drawn on the CDK4 image in panel ( A ). ( C ) Subcellular localization of cyclin D1, D2, and D3 (red) in wild-type HeLa cells. Scale bar, 10 μm. ( D , E ) The line-scan profile shows the fluorescence intensity across the membrane ruffles, cytoplasm, and nucleus along the line drawn on the cyclin D1/D2 images in panel ( C ). ( F ) Immunofluorescence staining showing co-localization of CDK4 (purple) and cyclin D1 (red) at the lamellipodia region. ‘Merge’ indicates the original merged image; ‘Merge (saturation-enhanced)’ shows the merged image after saturation adjustment to better visualize the co-localization of CDK4 with cyclin D1 within lamellipodia. Scale bar, 10 μm. ( G ) The linear intensity profile along the line shown in the Merge image of panel ( F ). Pearson’s correlation coefficient (PCC) R = 0.9333. ( H ) Immunofluorescence staining showing co-localization of CDK4 (purple) and cyclin D2 (red) at the lamellipodia region. Scale bar, 10 μm. ( I ) The intensity profile along the line shown in the Merge image of panel ( H ). PCC R = 0.8727.

Article Snippet: Cyclin D1 (Proteintech, Wuhan, China, 26939-1-AP, 1:500 for immunofluorescence (IF)), Cyclin D2 (Proteintech, Wuhan, China, 10934-1-AP, 1:500 for IF), Cyclin D3 (Proteintech, Wuhan, China, 26755-1-AP, 1:500 for IF), CDK4 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-23896, 1:500 for IF), CDK6 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7961, 1:500 for IF), Phospho-FAK (Tyr397) (Cell Signaling Technology, Danvers, MA, USA, 3283S, 1:1000 for immunoblotting (IB)), FAK (Cell Signaling Technology, Danvers, MA, USA, 3285S, 1:1000 for IB), Paxillin (Abcam, Waltham, MA, USA, ab32084, 1:50 for IF), GAPDH (Proteintech, Wuhan, China, 10494-1-AP, 1:5000 for IB).

Techniques: Staining, Fluorescence, Membrane, Immunofluorescence

Journal: Journal of Molecular Endocrinology

Article Title: Actions of 1,25(OH)2-vitamin D3 on the cellular cycle depend on VDR and p38 MAPK in skeletal muscle cells

doi: 10.1530/jme-14-0102

Figure Lengend Snippet: Figure 7 Silencing of cyclin D3 by a specific siRNA affects VDR and myogenin expression in muscle cells. C2C12 WT cells were transfected with 50 pmol cyclin D3 siRNA or control siRNA for 6 h according to the manufacturer’s specifications. Transfected cells were incubated for an additional 18 h period in fresh medium until treatments were administered (as indicated previously). Western blot analyses were carried out with anti-cyclin D3, anti-VDR, and anti-myogenin antibodies. The blotted membranes were reprobed with anti-tubulin antibody in order to ensure equal loading of gels (data not shown). Representative immunoblots of blots from two independent experiments are shown.

Article Snippet: Anti-myogenin (sc-12732), anti-VDR (sc-1008) and antitubulin antibodies (sc-73242), HRP-conjugated secondary antibodies (anti-rabbit, sc-2004, and anti-mouse, sc-2005), and siRNA against cyclin D3 mRNA (sc-35137) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Control, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Vav Is Required for Cyclin D2 Induction and Proliferation of Mouse B Lymphocytes Activated via the Antigen Receptor

doi: 10.1074/jbc.m105305200

Figure Lengend Snippet: FIG. 6. Inhibition of p38 MAPK represses BCR-mediated pro- liferation but not cyclin D2 induction. A, [3H]thymidine incorpora- tion was used to monitor S-phase entry in cultures of small dense B cells following treatment with monoclonal anti-IgM (10 g/ml b.7.6) for the indicated times in the presence (open squares) or absence (filled squares) of 10 M SB203580 or with no stimulation (circles). Each data point represents a quadruplicate mean ( S.D.). B, whole cell lysates from cells treated as described in A were prepared for Western blotting as described above.

Article Snippet: The antibodies against cyclin D2 (M-20), cyclin D3 (18B6– 10), cyclin E (M-20), CDK2 (M2), CDK4 (C-22), CDK6 (C-21), and p27 (C-19) were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Vav Is Required for Cyclin D2 Induction and Proliferation of Mouse B Lymphocytes Activated via the Antigen Receptor

doi: 10.1074/jbc.m105305200

Figure Lengend Snippet: FIG. 7. Inhibition of Ca2 flux abrogates BCR mediated prolif- eration and cyclin D2 induction. A, [3H]thymidine incorporation was used to monitor S-phase entry in cultures of small dense B cells following treatment with monoclonal anti-IgM (10 g/ml b.7.6) for the indicated times in the presence (open squares) or absence (filled squares) of 10 g/ml SK&F 96365, or with no stimulation (circles). Each data point represents a quadruplicate mean ( S.D.). B, whole cell lysates from cells treated as described in A were prepared for Western blotting as described above. C, whole cell lysates from cultures of small dense B cells following treatment with monoclonal anti-IgM (10 g/ml b.7.6) for 24 h in the presence or absence of 10 g/ml SK&F 96365 added 3 h after the addition of anti-IgM.

Article Snippet: The antibodies against cyclin D2 (M-20), cyclin D3 (18B6– 10), cyclin E (M-20), CDK2 (M2), CDK4 (C-22), CDK6 (C-21), and p27 (C-19) were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: A Proteomic Survey Indicates Sortilin as a Secondary Substrate of the ER Translocation Inhibitor Cyclotriazadisulfonamide (CADA) *

doi: 10.1074/mcp.M116.061051

Figure Lengend Snippet: Potential CADA-affected PowerBlot targets selected for validation

Article Snippet: Western blotting antibodies were purchased from (i) BD Biosciences: anti-human CD4 [clone SK3], GAP1m [clone 15], p19 skp1 [clone 52], sequestome-1/p62 Lck ligand [clone 3], FUS/TLS [clone 15], XRCC4 [clone 4], p56 Lck [clone 28], Rb2 [clone 10], clathrin [clone 23]; (ii) Thermo Scientific: anti-c-Jun [clone 5B1], STAT1 [clone 15H3], cyclin D3 [clone DCS-22]; (iii) R&D Systems: anti-sortilin [clone 334708]; (iv) Abcam: anti-sortilin [clone {"type":"entrez-protein","attrs":{"text":"EPR15010","term_id":"523380793","term_text":"EPR15010"}} EPR15010 ]; (v) Abnova: anti-ASAP-1 [clone 2G7]; (vi) Dako: HRP-labeled goat anti-mouse immunoglobulins, HRP-labeled swine anti-rabbit immunoglobulins; and (vii) Cell Signaling Technologies: polyclonal anti-LAT, pLAT (Tyr191), pZAP-70 (Tyr493), monoclonal anti ZAP-70 [clone D1C10E], pZAP-70 (Tyr319) [clone 65E4], HRP-labeled goat anti-rabbit immunoglobulins.

Techniques: Activity Assay, Ubiquitin Proteomics, Non-Homologous End Joining

Journal: Molecular Biology of the Cell

Article Title: Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

doi: 10.1091/mbc.E21-05-0255

Figure Lengend Snippet: GSK3β down-regulates cyclin D3 in KRT19 KO cells. (A) Colony formation assay was performed in parental (P) and KRT19 KO (KO) cells transiently transfected with vector control or cyclin D3. Colony area normalized to parental cells transfected with vector control is shown as mean ± SEM. N = 3. Bar, 5 mm. (B) Whole cell lysates of parental (P) and KRT19 KO (KO1 and KO2) cells treated with 10 nM MG132 for the indicated time periods were harvested, and immunoblotting was performed with antibodies against the indicated proteins. Signal intensities of cyclin D3 normalized to the α-tubulin loading control and 0 h controls are shown as mean ± SEM. N = 4. (C) Whole cell lysates of parental (P) and KRT19 KO (KO1 and KO2) cells treated with 10 mM LiCl (+) or DMSO vehicle control (–) for 8 h were harvested and immunoblotting was performed with antibodies against the indicated proteins. Signal intensities of cyclin D3 normalized to the α-tubulin loading control and DMSO controls are shown as mean ± SEM. N = 4. (D) Whole cell lysates of parental (P) and KRT19 KO (KO1 and KO2) cells transfected with GSK3β (3β) or scrambled (SCR) siRNA for 48 h were harvested, and immunoblotting was performed with antibodies against the indicated proteins. Signal intensities of cyclin D3 normalized to the α-tubulin loading control and SCR siRNA transfected controls are shown as mean ± SEM. N = 6. (E) Whole cell lysates of parental and KRT19 KO cells transfected with two different GSK3β (A and B) or scrambled (SCR) siRNA for 72 h were harvested, and immunoblotting was performed with antibodies against the indicated proteins. (F) MTT assays were performed on parental and KRT19 KO cells transfected with two different GSK3β (A and B) or scrambled (SCR) siRNA for 72 h. The absorbance at 570 nm of cells with GSK3β knockdown was normalized to that of its scrambled siRNA control to calculate cell viability. Cell viability normalized to SCR siRNA transfected controls are shown as mean ± SEM. N = 5. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant.

Article Snippet: Cyclin D3 cDNA was cloned out of Rc/CMV cyclin D3 (CMV promoter; #10912 from Addgene) with PCR using the primers listed in and cloned into pmCherry-C1 (CMV promoter; Takara Bio). pEGFP-C3 K19 WT, pEGFP-C3 K19 S10A, and pEGFP-C3 K19 S35A were generated using plasmids pMRB10-K19 WT, pMRB10-K19 S10A, and pMRB10-K19 S35A (courtesy of Bishr Omary [ Zhou et al ., 1999 ]) along with the primers listed in and the In-Fusion HD cloning system (Takara Bio, Mountain View, CA) following the manufacturer’s instructions.

Techniques: Colony Assay, Transfection, Plasmid Preparation, Control, Western Blot, Knockdown

Journal: Molecular Biology of the Cell

Article Title: Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

doi: 10.1091/mbc.E21-05-0255

Figure Lengend Snippet: Activating GSK3β facilitates cyclin D3 degradation in KRT19 KO cells. (A) Whole cell lysates of parental (P) and KRT19 KO (KO1 and KO2) cells treated with 100 µM forskolin for the indicated time periods were harvested, and immunoblotting was performed with antibodies against the indicated proteins. Signal intensities of cyclin D3 normalized to the α-tubulin loading control and 0 h controls are shown as mean ± SEM. N = 4. (B) KRT19 KO (KO2) cells stably expressing GFP or GFP-K19 were treated with 100 µM forskolin (+) or DMSO vehicle control (–) for 8 h, and whole cell lysates were harvested. Immunoblotting was performed with antibodies against the indicated proteins, and signal intensities of cyclin D3 normalized to the α-tubulin loading control and DMSO controls are shown as mean ± SEM. N = 5. (C) Whole cell lysates of parental (P) and KRT19 KO cells treated with 100 µM forskolin for the indicated time periods were harvested, and immunoblotting was performed with antibodies against the indicated proteins. (D) Parental (P) and KRT19 KO (KO1 and KO2) cells were pretreated with 20 ng/µl of cycloheximide (CHX) or DMSO vehicle control (–) for 30 min before being treated with 100 µM forskolin for the indicated time periods. Whole cell lysates were harvested, and immunoblotting was performed with antibodies against the indicated proteins. (E) Parental (P) and KRT19 KO (KO1 and KO2) cells were pretreated with 10 nM MG132 (+) or DMSO vehicle control (–) for 30 min before being treated with 100 μM forskolin (+) or DMSO (–) for an additional 8 h. Whole cell lysates were harvested, and immunoblotting was performed with antibodies against the indicated proteins. Signal intensities of cyclin D3 normalized to the α-tubulin loading control and DMSO controls are shown as mean ± SEM. N = 4. (F) Parental (P) and KRT19 KO (KO1 and KO2) cells were pretreated with 10 mM LiCl (+) or DMSO vehicle control (–) for 30 min before being treated with 100 μM forskolin (+) or DMSO (–) for an additional 8 h. Whole cell lysates were harvested, and immunoblotting was performed with antibodies against the indicated proteins. Signal intensities of cyclin D3 normalized to the α-tubulin loading control and DMSO controls are shown as mean ± SEM. N = 4. (G) Parental (P) and KRT19 KO (KO1 and KO2) cells were transfected with GSK3β (3β) or scrambled (SCR) siRNA for 48 h before being treated with 100 μM forskolin for 8 h. Whole cell lysates were harvested, and immunoblotting was performed with antibodies against the indicated proteins. Signal intensities of cyclin D3 normalized to the α-tubulin loading control and DMSO are shown as mean ± SEM. N = 5. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant.

Article Snippet: Cyclin D3 cDNA was cloned out of Rc/CMV cyclin D3 (CMV promoter; #10912 from Addgene) with PCR using the primers listed in and cloned into pmCherry-C1 (CMV promoter; Takara Bio). pEGFP-C3 K19 WT, pEGFP-C3 K19 S10A, and pEGFP-C3 K19 S35A were generated using plasmids pMRB10-K19 WT, pMRB10-K19 S10A, and pMRB10-K19 S35A (courtesy of Bishr Omary [ Zhou et al ., 1999 ]) along with the primers listed in and the In-Fusion HD cloning system (Takara Bio, Mountain View, CA) following the manufacturer’s instructions.

Techniques: Western Blot, Control, Stable Transfection, Expressing, Transfection

Journal: Molecular Biology of the Cell

Article Title: Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

doi: 10.1091/mbc.E21-05-0255

Figure Lengend Snippet: Impact of K19 on GSK3β and cyclin D3 localizations inside the cell. (A) Parental and KRT19 KO cells were treated with 100 µM forskolin (+) or DMSO vehicle control (–) for 8 h and then immunostained with anti-GSK3β antibodies. Images were obtained using a confocal microscope. Nuclei are shown with DAPI. Bar, 20 µm. (B) Subcellular fractionation of parental (P) and KRT19 KO cells treated with 100 μM forskolin (+) or DMSO vehicle control (–). Immunoblotting was performed with antibodies against the indicated proteins. PARP was used as a control for the nuclear fraction, whereas GAPDH was used for the cytoplasmic fraction. (C) Signal intensities of nuclear GSK3β from (B) normalized to the PARP loading control and DMSO controls are shown as mean ± SEM. N = 5. (D) Parental (P) and KRT19 KO (KO1 and KO2) cells were immunostained with anti-cyclin D3 antibody, and images were obtained using an epifluorescence microscope. Nuclei are shown with DAPI. Bar, 20 µm. (E) CTCF of cyclin D3 from (D) was quantitated and normalized to the background CTCF. Nuclear/cytoplasmic cyclin D3 levels normalized to the parental control are shown as scatter box plots with median ± maxima and minima; n = 18 cells for each cell line. (F) Subcellular fractionation of parental (P) and KRT19 KO (KO) cells. Immunoblotting was performed with antibodies against the indicated proteins. PARP was used as a control for the nuclear fraction, whereas GAPDH was used for the cytoplasmic fraction. (G) Signal intensities of cyclin D3 from F were quantitated. Nuclear/cytoplasmic ratios of cyclin D3 normalized to the parental control are shown as mean ± SEM. N = 4. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant.

Article Snippet: Cyclin D3 cDNA was cloned out of Rc/CMV cyclin D3 (CMV promoter; #10912 from Addgene) with PCR using the primers listed in and cloned into pmCherry-C1 (CMV promoter; Takara Bio). pEGFP-C3 K19 WT, pEGFP-C3 K19 S10A, and pEGFP-C3 K19 S35A were generated using plasmids pMRB10-K19 WT, pMRB10-K19 S10A, and pMRB10-K19 S35A (courtesy of Bishr Omary [ Zhou et al ., 1999 ]) along with the primers listed in and the In-Fusion HD cloning system (Takara Bio, Mountain View, CA) following the manufacturer’s instructions.

Techniques: Control, Microscopy, Fractionation, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

doi: 10.1091/mbc.E21-05-0255

Figure Lengend Snippet: Identification of K19 domains required for GSK3β interaction. (A) Schematics of K19 mutants. GFP was tagged at the N-terminus of K19 WT and mutants. HR contains GFP fused to H and R domains of K19; RT, GFP fused to R and tail domains; H, GFP fused to H domain alone; and R, GFP fused to R domain alone. (B) IP was performed with anti-GFP–conjugated beads in KRT19 KO cells transiently transfected with the GFP-K19 chimeras described in A or GFP control. IP and inputs were subjected to SDS–PAGE and immunoblotting was performed with antibodies against the indicated proteins. (C) KRT19 KO cells stably expressing GFP-K19 WT (WT), GFP-K19 RT (RT), or GFP control were treated with 100 μM forskolin (+) or DMSO vehicle control (–) for 10 min. Whole cell lysates were processed for triton solubility. Immunoblotting was performed with antibodies against the indicated proteins. (D) Signal intensities of GSK3β from (C) normalized to the actin loading control. Triton-insoluble/soluble GSK3β levels normalized to DMSO controls are shown as mean ± SEM. N = 4. (E) KRT19 KO cells stably expressing GFP-K19 WT (WT), GFP-K19 RT (RT), or GFP control were treated with 100 μM forskolin for the indicated time periods. Whole cell lysates were harvested, and immunoblotting was performed with the indicated antibodies. (F) Signal intensities of pGSK3β (Ser9) and GSK3β from E were quantitated and normalized to the α-tubulin loading control. Ratios of pGSK3β/GSK3β relative to 0′ controls are shown as mean ± SEM. N = 3. (G) IP was performed with anti-GFP–conjugated beads in KRT19 KO cells transiently transfected with the indicated GFP-K19 chimeras (WT or S10A, S35A, or Y4F mutants) or GFP control. IP and inputs were subjected to SDS–PAGE and immunoblotting was performed with antibodies against the indicated proteins. (H) Signal intensities of GSK3β/GFP IP from (G) normalized to that of WT are shown as mean ± SEM. N = 3. I) KRT19 KO cells transiently transfected with GFP-K19 chimeras (WT or S10A, S35A, or Y4F mutants) or GFP control were treated with 100 μM forskolin for 8 h. Whole cell lysates were harvested, and immunoblotting was performed with the indicated antibodies. (J) Signal intensities of cyclin D3 from (I) normalized to the α-tubulin loading control and DMSO controls are shown as mean ± SEM. N = 5. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant.

Article Snippet: Cyclin D3 cDNA was cloned out of Rc/CMV cyclin D3 (CMV promoter; #10912 from Addgene) with PCR using the primers listed in and cloned into pmCherry-C1 (CMV promoter; Takara Bio). pEGFP-C3 K19 WT, pEGFP-C3 K19 S10A, and pEGFP-C3 K19 S35A were generated using plasmids pMRB10-K19 WT, pMRB10-K19 S10A, and pMRB10-K19 S35A (courtesy of Bishr Omary [ Zhou et al ., 1999 ]) along with the primers listed in and the In-Fusion HD cloning system (Takara Bio, Mountain View, CA) following the manufacturer’s instructions.

Techniques: Transfection, Control, SDS Page, Western Blot, Stable Transfection, Expressing, Solubility

Journal: Molecular Biology of the Cell

Article Title: Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

doi: 10.1091/mbc.E21-05-0255

Figure Lengend Snippet: H domain of K19 is required to maintain proper cyclin D3 and GSK3β localization. (A) KRT19 KO cells stably expressing GFP-K19 WT (WT), GFP-K19 HR (HR), GFP-K19 (RT), or GFP control were immunostained with anti-cyclin D3 and anti-GFP antibodies. Images were obtained using an epifluorescence microscope. Nuclei are shown with DAPI. Bar, 20 µm. (B) CTCF of cyclin D3 from A was quantitated and normalized to the background CTCF. Nuclear/cytoplasmic cyclin D3 levels normalized to the GFP control are shown as scatter box plots with median ± maxima and minima; n = 31 cells for each cell line. (C) Subcellular fractionation of KRT19 KO cells stably expressing GFP-K19 WT (WT), GFP-K19 RT (RT), or GFP control. Immunoblotting was performed with antibodies against the indicated proteins. PARP was used as a control for the nuclear fraction, whereas α-tubulin was used for the cytoplasmic fraction. (D) Signal intensities of cyclin D3 from C were quantitated. Nuclear/cytoplasmic cyclin D3 levels normalized to the GFP controls are shown as mean ± SEM. N = 8. (E) Subcellular fractionation of KRT19 KO cells stably expressing GFP-K19 WT (WT), GFP-K19 RT (RT), or GFP control treated with 100 µM forskolin (+) or DMSO vehicle control (–). Immunoblotting was performed with antibodies against the indicated proteins. PARP was used as a control for the nuclear fraction, whereas α-tubulin was used for the cytoplasmic fraction. (F) Signal intensities of GSK3β from (E) normalized to the PARP loading control and DMSO controls are shown as mean ± SEM. N = 6. (G) KRT19 KO cells transiently transfected with GFP-K19 WT (WT), GFP-K19 S10A (S10A), GFP-K19 S35A (S35A), or GFP control were immunostained with anti-cyclin D3 and anti-GFP antibodies. Images were obtained using an epifluorescence microscope. Nuclei are shown with DAPI. Bar, 20 µm. (H) CTCF of cyclin D3 from G was quantitated and normalized to the background CTCF. Nuclear/cytoplasmic cyclin D3 levels normalized to the GFP control are shown as scatter box plots with median ± maxima and minima; n = 19 cells for each condition. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant.

Article Snippet: Cyclin D3 cDNA was cloned out of Rc/CMV cyclin D3 (CMV promoter; #10912 from Addgene) with PCR using the primers listed in and cloned into pmCherry-C1 (CMV promoter; Takara Bio). pEGFP-C3 K19 WT, pEGFP-C3 K19 S10A, and pEGFP-C3 K19 S35A were generated using plasmids pMRB10-K19 WT, pMRB10-K19 S10A, and pMRB10-K19 S35A (courtesy of Bishr Omary [ Zhou et al ., 1999 ]) along with the primers listed in and the In-Fusion HD cloning system (Takara Bio, Mountain View, CA) following the manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Control, Microscopy, Fractionation, Western Blot, Transfection

Journal: Molecular Biology of the Cell

Article Title: Keratin 19 interacts with GSK3β to regulate its nuclear accumulation and degradation of cyclin D3

doi: 10.1091/mbc.E21-05-0255

Figure Lengend Snippet: GSK3β inhibition sensitizes cells lacking K19 to CDK4/6 inhibitors. (A) Colony formation assay was performed in parental and KRT19 KO cells cultured for 3 d in the presence of ribociclib or palbociclib alone or in combination with or without CHIR 99021. Bar = 5 mm. (B) Colony area from (A) normalized to the parental control is shown as mean ± SEM. N = 6. (C) Colony formation assay was performed using KRT19 KO cells stably expressing GFP as control or GFP-K19 cultured for 3 d in the presence of ribociclib or palbociclib alone or in combination with or without CHIR 99021. Bar = 5 mm (D) Colony area from (C) normalized to the parental control is shown as mean ± SEM. N = 4. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, not significant. (E) A model of how K19 inhibits GSK3β-mediated degradation of cyclin D3. K19 binds to GSK3β and prevents its accumulation in the nucleus. K19–GSK3β interaction requires the R domain of K19 and activation of GSK3β. Ser10 and Ser35 of K19 also regulate K19–GSK3β interaction. Lack of K19 or introducing a GSK3β-binding deficient K19 mutant into cells increases nuclear GSK3β levels, leading to cyclin D3 degradation, decreased cell proliferation, and increased sensitivity to CDK4/6 inhibitors (CDKi).

Article Snippet: Cyclin D3 cDNA was cloned out of Rc/CMV cyclin D3 (CMV promoter; #10912 from Addgene) with PCR using the primers listed in and cloned into pmCherry-C1 (CMV promoter; Takara Bio). pEGFP-C3 K19 WT, pEGFP-C3 K19 S10A, and pEGFP-C3 K19 S35A were generated using plasmids pMRB10-K19 WT, pMRB10-K19 S10A, and pMRB10-K19 S35A (courtesy of Bishr Omary [ Zhou et al ., 1999 ]) along with the primers listed in and the In-Fusion HD cloning system (Takara Bio, Mountain View, CA) following the manufacturer’s instructions.

Techniques: Inhibition, Colony Assay, Cell Culture, Control, Stable Transfection, Expressing, Activation Assay, Binding Assay, Mutagenesis