Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 1. miR-206 targets cyclin D1. (A) Sequence alignment between miR-206 and the 3′UTRs of cyclin D1 from different species. In brackets the 3′UTR size. (B) Diagram of the luciferase reporter construct with the putative miR-206 binding site (WT 3′UTR) and mutations (3′UTR MUT). (C) Relative lucif- erase activity was measured in HeLa cells after transfection of reporter constructs along with pSP65-U1 (CTR) or pSP65–206 (miR-206). Relative Firefly luciferase values were determined by a ratio of Firefly to Renilla luciferase with the control set to 1.00. Values are the means ± SD of 3 separate experi- ments. **A Student t test performed between control and miR-206 transfected cells yielded P values < 0.01.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Sequencing, Luciferase, Construct, Binding Assay, Activity Assay, Transfection, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 2. Expression kinetics of miR-206 and cyclin D1 in differentiating C2C12 cells. C2C12 myoblasts were seeded in GM at 1.5 × 104/cm2. Cells were shifted in DM 24 h after plating and left to differentiate for further 72 h. (A) Northern blot analysis of miR-206 expression in C2C12 cells after 24 h in GM (0) and at different time points upon shift to DM. (B) Western blot analysis of cyclin D1 and MyHC expression in C2C12 cells cultured as in (A). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) MyHC immunofluorescence staining (green) of C2C12 cells after 24 h in GM (DM 0 h) and after 72 h in DM (DM 72 h). Nuclei were counterstained in blue (DAPI) and individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 20 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Western Blot, Cell Culture, Immunofluorescence, Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 3. miR-206 controls cyclin D1 accumulation in C2C12 cells. C2C12 myoblasts were seeded in GM at 2.5 × 103/cm2. Cells were transfected 24 h after plaiting. (A) Northern blot analysis of miR-206 expression (upper) and western blot analysis of cyclin D1 expression (lower) in C2C12 cells 48 h after transfection with a control vector (CTR) or with a miR-206 expression vector (miR-206). Cells were kept in GM throughout the experiment. (B) The effect of miR-206 overexpression on C2C12 cell proliferation and differentiation was evaluated 48 h after transfection by 1 h BrdU incorporation and MyHC staining, respectively. Results are represented relative to the BrdU+ nuclei or nuclei in MyHC+ cells in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. (C) Immunofluorescence staining of cyclin D1 (pink) and MyHC (green) 48 h after transfection. Nuclei were counterstained in blue with DAPI. Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. To obtain cyclin D1 images, before merging, individual pictures were pseudocol- ored using a LEICA Microsystems Imaging software. Bar = 10 μm. (D) C2C12 myoblasts were seeded at low (LD) and high (HD) density in GM. Cells were shifted to DM the day after plating and analyzed after further 3 d. The panels show a northern blot analysis of miR-206 expression (left panel) and a western blot analysis of cyclin D1 and differentiation associ- ated marker expression (right panel) after 24 h in GM and 72 h after shift- ing to DM. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Northern Blot, Expressing, Western Blot, Control, Plasmid Preparation, Over Expression, BrdU Incorporation Assay, Staining, Immunofluorescence, Imaging, Software, Marker

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 4. Inhibition of miR-206 rescues cyclin D1 in myotubes (A) Experimental scheme. C2C12 myoblasts were induced to differ- entiate in DM in the presence of AraC. After 3 d, AraC was washed out and cells left to recover in DM for further 24 h. Finally, pure myotubes were transfected with LNA against miR-206 and analyzed 48 h later. (B) Northern blot analysis of miR-206 and miR-1 expression (left panel) and western blot analysis of cyclin D1 expression (right panel) in pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Cyclin D1 expression in proliferating myoblasts is also shown (GM). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) Double immunofluorescence staining of MyHC and cyclin D1 of pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 10 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Inhibition, Transfection, Northern Blot, Expressing, Western Blot, Control, Double Immunofluorescence Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 5. miR-206 inhibits cell proliferation in Ras-transformed fibro- blasts. (A) Expression levels of cyclin D1 in NIH3T3(Ras) cells as compared with NIH3T3(BN) cells. (B) Real-time PCR analysis of miR-206 expres- sion in NIH3T3(Ras) cells. Results are shown relative to untransformed NIH3T3(BN) cells set to value 1.00. Each sample was analyzed in tripli- cate, and values are the means ± SD of 3 independent experiments. **A Student t test performed between untransformed and transformed cells yielded P values < 0.01. (C) NIH3T3(Ras) cells were transfected with a control vector (CTR) or with a miR-206 expression vector (miR-206) and analyzed 24 h later. Upper, northern blot analysis of miR-206 expression; lower, western blot analysis of cyclin D1 expression. (D) Effect of miR-206 forced expression on cell proliferation as determined by 1 h BrdU incor- poration. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. Equal RNA and protein loading was confirmed by detecting, snRNA U2, and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 6. Relationship between miR-206 downregulation and cyclin D1 expression in NSCLCs. (A) Northern blot analysis of miR-206 in different murine tissues. snRNA U2 levels were used as a loading control. (B) Real-time PCR analysis of miR-206 expression in human NSCLC tissues. Results are shown relative to the matched normal lung tissues set to value 1.00. Each sample was analyzed in triplicate, and values are the means ± SD of three independent experiments. **A Student t test performed between normal and tumor tissues yielded P values < 0.01. (C) Western blot analysis of cyclin D1 expression in normal and neoplastic lung tissues. Equal protein loading was confirmed by detecting actin. n, normal tissue; t = tumor tissue

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 7. miR-206 inhibits cancer cell proliferation through repression of cyclin D1. (A) A549 and HeLa cells were transfected with a control vec- tor (CTR) or with a miR-206 expression vector (miR-206) and analyzed 72 h later. Top panel: northern blot analysis of miR-206 expression; lower panel, western blot analysis of cyclin D1 expression. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (B) Effect of miR-206 forced expression on cell prolifera- tion as determined by 1 h BrdU incorporation and immunofluorescence staining. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values <0.05.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Northern Blot, Western Blot, BrdU Incorporation Assay, Immunofluorescence, Staining

Journal: Aging (Albany NY)

Article Title: Restoration of aged hematopoietic cells by their young counterparts through instructive microvesicles release

doi: 10.18632/aging.203689

Figure Lengend Snippet: Key Resources Table.

Article Snippet: Anti-Human Cyclin D1 (WB) , Cell Signaling , Cat #2978.

Techniques: Recombinant, Saline, Protease Inhibitor, SYBR Green Assay, Isolation, Cytotoxicity Assay, Viability Assay, Software, Expressing, RNA Sequencing, Sequencing, Real-time Polymerase Chain Reaction

Journal: Molecules

Article Title: Structural Optimization and Improving Antitumor Potential of Moreollic Acid from Gamboge

doi: 10.3390/molecules27020482

Figure Lengend Snippet: TH12-10 blocks S phase entry. ( A ) Immunoblot analysis of Cyclin D1, CDK4, CDK6, and p-Rb in SW620 and DLD1 cells treated with indicated concentrations of TH12-10 for 48 h. ( B ) Densitometry analysis of indicated proteins as described in ( A ) was performed using Image J software. ( C ) Flow cytometric analysis for cell-cycle distribution in DLD1 cells treated with TH12-10 or vehicle and then with nocodazole for 48 h. ( D ) Quantitative analysis of DLD1 cells in G1 phase as described in ( C ). All data are presented as means ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. N.S., not significant. Differences were tested using one-way ANOVA ( B ) or the unpaired t-test with Welch’s correction ( D ).

Article Snippet: Antibodies used for the immunoblot analysis in this study were as follows: Cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), CDK4 (ab199728, Abcam, Cambridge, UK), CDK6 (13331T, Cell Signaling Technology, Danvers, MA, USA), phospho-Rb (Ser807/811) (8516S, Cell Signaling Technology, Danvers, MA, USA), and β-actin (sc-47778, Santa Cruz Biotechnology, CA, USA)

Techniques: Western Blot, Software

Journal: Molecules

Article Title: Structural Optimization and Improving Antitumor Potential of Moreollic Acid from Gamboge

doi: 10.3390/molecules27020482

Figure Lengend Snippet: The contribution of TH12-10 to the cell cycle. ( A ) Western blotting shows Cyclin D1, CDK4, CDK6, and p-Rb levels in NCM460 cells treated with TH12-10 at indicated concentrations for 48 h. ( B ) Densitometry analysis of indicated proteins as described in ( A ) was conducted and presented. ( C ) Flow cytometric analysis for cell-cycle distribution in NCM460 cells treated with TH12-10 or vehicle and then with nocodazole for a total of 48 h. ( D ) Quantitative analysis of NCM460 cells in G1 phase as described in ( C ). All data are presented as means ± SD of three independent experiments. N.S., not significant. Differences were tested using one-way ANOVA ( B ) or the unpaired t-test with Welch’s correction ( D ).

Article Snippet: Antibodies used for the immunoblot analysis in this study were as follows: Cyclin D1 (55506T, Cell Signaling Technology, Danvers, MA, USA), CDK4 (ab199728, Abcam, Cambridge, UK), CDK6 (13331T, Cell Signaling Technology, Danvers, MA, USA), phospho-Rb (Ser807/811) (8516S, Cell Signaling Technology, Danvers, MA, USA), and β-actin (sc-47778, Santa Cruz Biotechnology, CA, USA)

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Leucyl-tRNA synthetase deficiency systemically induces excessive autophagy in zebrafish

doi: 10.1038/s41598-021-87879-4

Figure Lengend Snippet: Inhibition of autophagy prevents abnormal development and improves survival in larsb- knockout larvae. (A) Morphology of larsb + / + and larsb −/− embryos injected with either control MO or atg5-MO (72 h post fertilization (hpf)). Scale bars: 500 µm. (B) Western blot analysis of Lc3b protein expression at 72 hpf for wild-type embryos injected with either control MO or atg5-MO. β-actin levels served as the loading control. (C) Morphological abnormality at 72 hpf in the livers of larsb −/− larvae under Tg[ fabp10 :mcherry] background injected with either control MO or atg5-MO. Scale bars: 200 μm. (D) Quantification of liver size in larsb −/− larvae under Tg[ fabp10 :mcherry] background (72 hpf). Liver sizes were evaluated using ImageJ software version 1.52a ( https://imagej.nih.gov/ij/ ). n = 4 fish/group. Error bars indicate SEM. Student’s t-test; ***P < 0.001. (E) Western blot analysis of Lc3b protein expression at 72 hpf for wild-type embryos treated with DMSO or bafilomycin A1. β-actin levels served as the loading control. (F) Morphological abnormality at 72 hpf in the livers of larsb −/− larvae under Tg[ fabp10 :mcherry] background treated with DMSO or bafilomycin A1. Scale bars: 200 μm. (G) Quantification of liver size in larsb −/− larvae under Tg[ fabp10 :mcherry] background (72 hpf). Liver sizes were evaluated using ImageJ software version 1.52a ( https://imagej.nih.gov/ij/ ). n = 10 fish/group. Error bars indicate SEM. Student’s t-test; *P < 0.05. (H) Kaplan–Meier survival curve of larsb + / + (n = 23) and larsb −/− (n = 15) larvae treated with DMSO and larsb + / + (n = 11) and larsb −/− larvae (n = 21) treated with bafilomycin A1. Statistics were calculated and the figure was produced in GraphPad software version 8 ( https://www.graphpad.com/scientific-software/prism/ ). Larsb: leucyl-tRNA synthetase b, MO: morpholino, n.s.: non-significant, DMSO: dimethyl sulfoxide, Dpf: days post fertilization.

Article Snippet: Western blotting was performed with antibodies against Lars (#13868; Cell Signaling Technology, Beverly, MA, USA), p62 (PM045; Medical & Biological Laboratories, Nagoya, Japan), LC3B (PM036; Medical & Biological Laboratories), ATG5 (NB110-53818; Novus Biologicals, Littleton, CO, USA), β-actin (A3854; Sigma-Aldrich, St. Louis, MO, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G9295; Sigma-Aldrich).

Techniques: Inhibition, Knock-Out, Injection, Control, Western Blot, Expressing, Software, Produced

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Down-regulation of dihydrofolate reductase inhibits the growth of endothelial EA.hy926 cell through induction of G1 cell cycle arrest via up-regulating p53 and p21 waf1/cip1 expression

doi: 10.3164/jcbn.15-64

Figure Lengend Snippet: Effect of DHFR knockdown on the expression of CDK2, CDK4, CDK6, cyclins D1 and E. At 72 h post-transfection with siRNA, cells were lysed with RIPA lysis buffer, and the lysates were then analyzed by western blot with specific antibodies (dilution 1:800). β-actin served as internal loading control (dilution 1:8,000). (A) Representative gels from 3 experiments are shown as protein expression levels of cyclin-dependent kinases CDK2, CDK4 and CDK6, as well as cyclins D1 and E. (B) Densitometric analysis was performed for each protein band relative to β-actin in the same sample using Image Lab TM software. To compare the relative quantity (RQ) of proteins between NC-siRNA and DHFR-siRNA groups, the values of density ratio in NC-siRNA group were normalized to 1.00. *vs NC-siRNA, p <0.05; **vs NC-siRNA, p <0.01.

Article Snippet: Primary antibodies for detecting DHFR (15194-1-AP), β actin (60008-1-Ig), CDK2 (10122-1-AP), CDK4 (11026-1-AP), CDK6 (14052-1-AP), Cyclin D1 (60186-1-Ig), Cyclin E (11554-1-AP), and p21 waf/cip1 (10355-1-AP) were purchased from Proteintech (Chicago, IL).

Techniques: Expressing, Transfection, Lysis, Western Blot, Software

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: Cell Culture, Transfection, Control, WST-1 Assay, Western Blot, Knockdown, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Overcoming AC0010 resistance by targeting c-MET in in vivo xenograft model. (A) Inhibition of H1975-P1-R1 tumor growth by AC0010 or crizotinib, alone or in combination, for a period of 14 days. Tumor growth curves are plotted as mean ± SEM ( n = 8). (B) Tumor weights of each group were analyzed using SPSS 17.0 software; t test statistical analysis. (C) Mouse body weights of each group are plotted as mean ± SEM ( n = 8). (D) H1975-P1-R1 tumor issues from mice treated with vehicle, AC0010, crizotinib, or AC0010 combined with crizotinib for 14 days were isolated and subjected to Western blotting analysis using indicated Abs.

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: In Vivo, Inhibition, Software, Isolation, Western Blot

Journal: iScience

Article Title: SGMS1 facilitates osteogenic differentiation of MSCs and strengthens osteogenesis-angiogenesis coupling by modulating Cer/PP2A/Akt pathway

doi: 10.1016/j.isci.2024.109358

Figure Lengend Snippet:

Article Snippet: Akt Mouse monoclonal antibody , Proteintech , 60203-2-Ig; RRID:AB_2919902.

Techniques: Recombinant, Virus, Modification, Reverse Transcription, SYBR Green Assay, IP Phosphatase Assay, Software