Journal: BMC Complementary and Alternative Medicine

Article Title: Anticancer activity of calyx of Diospyros kaki Thunb. through downregulation of cyclin D1 via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells

doi: 10.1186/s12906-017-1954-2

Figure Lengend Snippet: The effect of DKC-E70 on the expression of cyclin D1 at the protein and mRNA level in human colorectal cancer cells. The cells were plated overnight and then treated with DKC-E70 at the indicated concentrations for 24 h. For Western blot analysis ( a ), cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. Actin was used as internal control for Western blot analysis. For RT-PCR analysis of the gene expression of cyclin D1 ( b ), total RNA was prepared. GAPDH was used as internal control for RP-PCR. ( c ) HCT116 cells were treated with DKC-E70 (50 μg/ml) for the indicated times. The protein and mRNA level of cyclin D1 was analyzed using Western blot and RT-PCR, respectively. *P < 0.05 compared to cell without DKC-E70 treatment

Article Snippet: Wild type HA-tagged cyclin D1 and point mutation of T286A of HA-tagged cyclin D1 were provided from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Western Blot, SDS Page, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Complementary and Alternative Medicine

Article Title: Anticancer activity of calyx of Diospyros kaki Thunb. through downregulation of cyclin D1 via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells

doi: 10.1186/s12906-017-1954-2

Figure Lengend Snippet: DKC-E70 mediates cyclin D1 degradation. a HCT116 cells were pretreated with MG132 at the indicated concentration for 2 h, and then co-treated with DKC-E70 (50 μg/ml) for 10 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. b HCT116 ells were pretreated with DMSO or DKC-E70 (50 μg/ml), and then co-treated with 10 μg/ml of cycloheximide (CHX) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1.Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without DKC-E70 treatment

Article Snippet: Wild type HA-tagged cyclin D1 and point mutation of T286A of HA-tagged cyclin D1 were provided from Addgene (Cambridge, MA, USA).

Techniques: Concentration Assay, SDS Page, Western Blot

Journal: BMC Complementary and Alternative Medicine

Article Title: Anticancer activity of calyx of Diospyros kaki Thunb. through downregulation of cyclin D1 via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells

doi: 10.1186/s12906-017-1954-2

Figure Lengend Snippet: Cyclin D1 degradation by DKC-E70 is dependent on the activation of ERK1/2, p38 and GSK3β. a - c HCT116 cells were pretreated with PD98059 as an ERK1/2 inhibitor ( a ), SB203580 as a p38 inhibitor ( b ) or LiCl as a GSK3β inhibitor ( c ), and then co-treated with DKC-E70 (50 μg/ml) for 10 h. d HCT116 cells were treated with DKC-E70 (50 μg/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1, p-ERK1/2, total-ERK1/2, p-p38, total-p38, p-GSK3β or total-GSK3β. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without DKC-E70 treatment

Article Snippet: Wild type HA-tagged cyclin D1 and point mutation of T286A of HA-tagged cyclin D1 were provided from Addgene (Cambridge, MA, USA).

Techniques: Activation Assay, SDS Page, Western Blot

Journal: BMC Complementary and Alternative Medicine

Article Title: Anticancer activity of calyx of Diospyros kaki Thunb. through downregulation of cyclin D1 via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells

doi: 10.1186/s12906-017-1954-2

Figure Lengend Snippet: Cyclin D1 degradation by DKC-E70 is followed by T286 phosphorylation dependent on ERK1/2, p38 and GSK3β. a HCT116 cells were treated with DKC-E70 (50 μg/ml) for the indicated times. b HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector for 24 h, and then treated with DKC-E70 (50 μg/ml) for 10 h. c HCT116 cells were pretreated with PD98059 (40 μM) as an ERK1/2 inhibitor, SB203580 (40 μM) as a p38 inhibitor or LiCl (20 mM) as a GSK3β inhibitor, and then co-treated with DKC-E70 (50 μg/ml) for 3 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against p-cyclin D1 (T286) or HA-cyclin D1. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without DKC-E70 treatment. d HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector for 24 h, and then treated with DKC-E70 (50 μg/ml) for 24 h. Cell viability was measured using MTT assay. *P < 0.05 compared to cell without DKC-E70

Article Snippet: Wild type HA-tagged cyclin D1 and point mutation of T286A of HA-tagged cyclin D1 were provided from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, MTT Assay

Journal: BMC Complementary and Alternative Medicine

Article Title: Anticancer activity of calyx of Diospyros kaki Thunb. through downregulation of cyclin D1 via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells

doi: 10.1186/s12906-017-1954-2

Figure Lengend Snippet: Decreased level of cyclin D1 mRNA by DKC-E70 is attributed to the inhibition of Wnt activation. a HCT116 cells were treated with DKC-E70 at the indicated concentrations for 24 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against β-catenin or TCF4. Actin was used as internal control for Western blot analysis. b HCT116 cells were co-transfected with TOP-FLASH or FOP-FLASH constructs containing wild-type or mutated TCF binding sites and pRL-null. The cells were treated with DKC-E70 for 24 h. Luciferase activity for TOP-FLASH and FOP-FLASH was measured as a ratio of firefly luciferase signal/renilla luciferase signal using a dual luciferase assay kit

Article Snippet: Wild type HA-tagged cyclin D1 and point mutation of T286A of HA-tagged cyclin D1 were provided from Addgene (Cambridge, MA, USA).

Techniques: Inhibition, Activation Assay, SDS Page, Western Blot, Transfection, Construct, Binding Assay, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination

doi: 10.1074/jbc.m110.164384

Figure Lengend Snippet: FIGURE 1. Correlation between SLUG and cyclin D1 levels in breast tis- sues and cells. A, human breast cancer tissue microarray was examined for cyclin D1 and SLUG expressions by immunohistochemistry using mouse anti-cyclin D1 and rabbit anti-SLUG antibodies. A low magnification (4) immunofluorescence micrograph of the array is shown in the supplemental material (supplemental Fig. 1S). We show here a higher magnification (30) immunofluorescence micrograph for selected spots. The coordinates of the spots are marked as detailed in supplemental Table 3S. B, evaluation of the levels of cyclin D1 in normal and SLUG-expressing human breast cancer cells by immunofluorescence microscopy. 468C and MCF7C, MDA-MB-468 and MCF7 cells transfected with empty vector; 468SLUG and MCF7SLUG, cells expressing C-terminal FLAG-tagged SLUG; 231C and BT549C, normal MDA-MB-231 and BT549 cells.

Article Snippet: Wild-type human cyclin D1 (in pCMV; Addgene plasmid 19927) (35) and the T286A mutant of human cyclin D1 (C-terminally HA-tagged in pCDNA3.0; Addgene plasmid 11182) (36) as well as the N-terminally HA-tagged ubiquitin expression plasmid (in pcDNA3.1; Addgene plasmid 18712) (37) were procured from Addgene Inc. (Cambridge, MA).

Techniques: Microarray, Immunohistochemistry, Immunofluorescence, Expressing, Microscopy, Transfection, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination

doi: 10.1074/jbc.m110.164384

Figure Lengend Snippet: FIGURE 2. Effect of SLUG overexpression on the levels of cyclin D1 in MDA-MB-468 cells. A, increase in cyclin D1 levels in SLUG-expressing MDA- MB-468 cells. A Western blot shows higher levels of cyclin D1 in the recom- binant cells. B, densitometric scan for cyclin D1 and SLUG levels in six inde- pendent SLUG-transfected populations and vector controls. Results are mean S.E. (error bars) (n 6). The -fold changes were statistically signifi- cant (p 0.001). C, evaluation of SLUG, cyclin D1, and UbcH5c protein levels in four different breast cancer cell lines by Western blotting. Bands were developed using IR dye-conjugated secondary antibody (LI-COR Bio- sciences) and visualized using the LI-COR Odyssey infrared imaging system. Quantitation and analysis of bands were performed using Odyssey soft- ware. -Actin was used as normalization control. Levels of SLUG and cyclin D1 in the MDA-MB-231 cells and that of UbcH5c in the MCF7 cells were taken as 100 for comparison. Results are mean S.E. (n 4).

Article Snippet: Wild-type human cyclin D1 (in pCMV; Addgene plasmid 19927) (35) and the T286A mutant of human cyclin D1 (C-terminally HA-tagged in pCDNA3.0; Addgene plasmid 11182) (36) as well as the N-terminally HA-tagged ubiquitin expression plasmid (in pcDNA3.1; Addgene plasmid 18712) (37) were procured from Addgene Inc. (Cambridge, MA).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Imaging, Quantitation Assay, Control, Comparison

Journal: Journal of Biological Chemistry

Article Title: SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination

doi: 10.1074/jbc.m110.164384

Figure Lengend Snippet: FIGURE 3. Effect of SLUG expression on UbcH5c and cyclin D1 levels in MDA-MB-468 and MCF7 cells. A, a typical immunoblot showing UbcH5c and SLUG protein levels in different human breast cancer cells. MB468, MDA-MB-468 cells; MB231, MDA-MB-231 cells. B, real-time RT-PCR analysis of the levels of SLUG, UbcH5c, and cyclin D1 mRNAs in SLUG-overexpressing MCF7 and MDA-MB-468 cells. Results are mean S.E. (n 6). The differences between the experimental and control sets were statistically significant (p 0.001). C, immunoblot analysis for SLUG and UbcH5c proteins in the control and SLUG-over- expressing (SLUG) MCF7 and MDA-MB-468 cells. D, densitometric scan for SLUG and UbcH5c levels in six independent SLUG-transfected populations and corresponding vector-transfected control cells. Results are mean S.E. (n 6). The -fold changes were statistically significant (p 0.001). E, immunoblot analysis data showing the effects of SLUG overexpression on the levels of cyclin D1, phosphocyclin D1 (at Thr286), GSK3, phospho-GSK3, AKT, and phos- pho-AKT in MDA-MB-468 cells. Control cells (V) were transfected with empty vector DNA instead of SLUG construct plasmid DNA. Recombinant SLUG was FLAG-tagged at the C-terminal end and thus was detected with anti-FLAG antibody. -Actin was used as a loading control. F, effect of cyclin D1 knockdown (CD1KD) on the SLUG-induced increase in cell proliferation and tamoxifen (4HT; 10 M) resistance in MCF7 cells. Control cells were transfected with empty vector DNA instead of SLUG construct plasmid DNA. Results are mean S.E. (n 6). Data with cyclin D1 siRNA stealth-311 (supplemental Table 2S) are shown. Other siRNA, stealth-568, also yielded similar results (data not shown). The ability of stealth-311 to knock down cyclin D1 in MDA-MB-231 cells is shown in supplemental Fig. 3S.

Article Snippet: Wild-type human cyclin D1 (in pCMV; Addgene plasmid 19927) (35) and the T286A mutant of human cyclin D1 (C-terminally HA-tagged in pCDNA3.0; Addgene plasmid 11182) (36) as well as the N-terminally HA-tagged ubiquitin expression plasmid (in pcDNA3.1; Addgene plasmid 18712) (37) were procured from Addgene Inc. (Cambridge, MA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Transfection, Plasmid Preparation, Over Expression, Construct, Recombinant, Knockdown

Journal: Journal of Biological Chemistry

Article Title: SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination

doi: 10.1074/jbc.m110.164384

Figure Lengend Snippet: FIGURE 4. Effect of knockdown of SLUG on cyclin D1 levels in MDA-MB- 231 and BT549 cells. A, quantitative RT-PCR analysis for SLUG, UbcH5c, and cyclin D1 mRNA levels in MDA-MB-231 cells treated with different siRNAs (supplemental Table 2S). B, immunoblot analysis of UbcH5c and cyclin D1 levels in MDA-MB-231 and BT549 cells with (SLUGKD) or without (Control) knocking down SLUG (siRNA#1, stealth-21). Control cells were transfected with control siRNA. C, evaluation of SLUG, cyclin D1, and UbcH5c protein levels in MDA-MB-231 and BT549 cells with or without knockdown of SLUG. Six independent SLUG-knocked down cell populations and corresponding control siRNA-treated cells were used. Data with siRNA#1 as the reagent for the knockdown are shown. Similar results were obtained with stealth-223 (siRNA#2; data not shown). Bands were developed using IR dye-conjugated secondary antibody (LI-COR Biosciences) and visualized using the LI-COR Odyssey infrared imaging system. Quantitation and analysis of bands were performed using Odyssey software. -Actin was used as normalization con- trol. Results are mean S.E. (error bars) (n 6). -Fold changes observed were statistically significant (p 0.001). D, effect of knockdown of SLUG in MDA-MB-231 cells on their rate of proliferation and the role of non-degrad- able cyclin D1 mutant (T286A) in this process. Cells were transiently trans- fected with wild type or HA-tagged T286A mutant of cyclin D1 along with the siRNA (control or anti-SLUG). Results are mean S.E. (n 6). The effect of SLUG knockdown with wild-type cyclin D1 was statistically significant (p 0.0001). E, effect of the proteasome inhibitor MG132 on the decrease in cyclin D1 in SLUG-knocked down MDA-MB-231 cells. Control cells were transfected with empty vector DNA. -Actin was used as loading control. Veh, vehicle (DMSO) for MG132 solution. F, densitometric analysis of immu- noblot data as in E from three independent experiments. The upper panel (i) shows the effect on SLUG level, and the lower panel (ii) shows the effect on cyclin D1 levels. Experimental data are normalized assuming respective control as 100. Results are mean S.E. (n 3); *, statistical significance in comparison with respective control (p 0.001).

Article Snippet: Wild-type human cyclin D1 (in pCMV; Addgene plasmid 19927) (35) and the T286A mutant of human cyclin D1 (C-terminally HA-tagged in pCDNA3.0; Addgene plasmid 11182) (36) as well as the N-terminally HA-tagged ubiquitin expression plasmid (in pcDNA3.1; Addgene plasmid 18712) (37) were procured from Addgene Inc. (Cambridge, MA).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Control, Transfection, Imaging, Quantitation Assay, Software, Mutagenesis, Plasmid Preparation, Comparison

Journal: Journal of Biological Chemistry

Article Title: SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination

doi: 10.1074/jbc.m110.164384

Figure Lengend Snippet: FIGURE 5. Effect of knockdown of UbcH5c on cyclin D1 levels in MDA- MB-468 and MCF7 cells. A, immunoblot analysis showing the effect of knockdown of UbcH5c (H5cKD) on cyclin D1 level in MDA-MB-468 cells. Con- trol cells were transfected with control siRNA. B, immunoblot analysis show- ing the effect of knockdown of UbcH5c on cyclin D1 level in MCF7 cells. -Actin was used as loading control. Control cells were transfected with control siRNA. C, evaluation of cyclin D1 and UbcH5c protein levels in MDA- MB-468 cells with or without knockdown of UbcH5c. Six independent UbcH5c-knocked down cell populations and corresponding control siRNA-treated cells were used. D, evaluation of cyclin D1 and UbcH5c pro- tein levels in MCF7 cells with or without knockdown of UbcH5c. Six inde- pendent UbcH5c-knocked down cell populations and corresponding con- trol siRNA-treated cells were used. For the experiments in C and D, bands were developed using IR dye-conjugated secondary antibody (LI-COR Bio- sciences) and visualized using the LI-COR Odyssey infrared imaging system. Quantitation and analysis of bands were performed using Odyssey soft- ware. -Actin was used as normalization control. Results are mean S.E. (error bars) (n 6). Fold changes observed were statistically significant (p 0.001). E, proliferation assays with the control and UbcH5c knockdown MCF7 cells in the absence or presence of 4HT (10 M) and the effects of si- multaneous knockdown of cyclin D1 (CD1H5cKD) in these processes. Re- sults are mean S.E. (n 6). Data with cyclin D1 siRNA stealth-311 (supple- mental Table 2S) are shown. Other siRNA, stealth-568, also yielded similar results (data not shown). F, Matrigel invasion assay with UbcH5c knocked down MCF7 and MDA-MB-468 cells. Results are mean S.E. (n 6). Data with siRNA stealth-1106 (supplemental Table 2S) are shown. Other siRNA, stealth-1214, also yielded similar results (data not shown).

Article Snippet: Wild-type human cyclin D1 (in pCMV; Addgene plasmid 19927) (35) and the T286A mutant of human cyclin D1 (C-terminally HA-tagged in pCDNA3.0; Addgene plasmid 11182) (36) as well as the N-terminally HA-tagged ubiquitin expression plasmid (in pcDNA3.1; Addgene plasmid 18712) (37) were procured from Addgene Inc. (Cambridge, MA).

Techniques: Knockdown, Western Blot, Transfection, Control, Imaging, Quantitation Assay, Invasion Assay

Journal: Journal of Biological Chemistry

Article Title: SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination

doi: 10.1074/jbc.m110.164384

Figure Lengend Snippet: FIGURE 6. Effect of overexpression of UbcH5c on cyclin D1 levels and proliferation in SLUG-high MDA-MB-231 cells. A, immunoblot analysis showing the effect of overexpression of UbcH5c on cyclin D1 level. Control cells were transfected with empty vector DNA. UbcH5c levels were assessed by using Myc antibody for the recombinant protein and UbcH5c antibody for the total of the endogenous and recombinant protein. B, proliferation assays with the control and UbcH5c-overexpressed (UbcH5cOE) MDA-MB- 231 cells. Results are mean S.E. (error bars) (n 6).

Article Snippet: Wild-type human cyclin D1 (in pCMV; Addgene plasmid 19927) (35) and the T286A mutant of human cyclin D1 (C-terminally HA-tagged in pCDNA3.0; Addgene plasmid 11182) (36) as well as the N-terminally HA-tagged ubiquitin expression plasmid (in pcDNA3.1; Addgene plasmid 18712) (37) were procured from Addgene Inc. (Cambridge, MA).

Techniques: Over Expression, Western Blot, Control, Transfection, Plasmid Preparation, Recombinant

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: Cyclin D1 enriched in EVs during neurogenesis. (A) Immunoblot analysis of cyclin D1, 2, and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1–D9, PC12 cells incubated with NGF for 1–9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0) and EVs purified from NGF-induced PC12 cells for 3, 6, and 9 d (D3, D6, and D9). (C) Quantitative immunoblot analysis of protein levels described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; **, P < 0.01). Error bars represent SD from independent samples. (D) Immunoblots of cyclin D1, Flot2, and Alix in EVs from undifferentiated ESCs (ES D0-EV) or 8-d (ES D8-EV) or 12-d (ES D12-EV) differentiated ESCs. (E) Immunoblots of cyclins, CDKs, Flot2, GM130, and actin in EVs and whole-cell lysates of PC12 cells or NGF-induced PC12 cells. (F) Immunoblot analysis of cyclin D1/2 and multiple EV markers of N6-EVs treated with different concentrations of proteinase K (PK), with or without 1% Triton X-100. (G) Immunoblots for cycinD1, Alix, Hsc70, Tsg101, and CD9 after immunoprecipitation of 5 × 10 10 N6-EV with anti-CD9 antibody. IP, immunoprecipitates.

Article Snippet: The cyclin D1–APEX plasmids were constructed by combining the PCR fragment of cyclin D1 from the cyclin D1–Flag plasmid and APEX from pcDNA3 APEX-nes (49386; Addgene) into XPack CMV constructs (System Biosciences).

Techniques: Western Blot, Incubation, Purification, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: Cyclin D1 is enriched in EVs during N2A neurogenesis. (A) Immunoblots of cyclin D, CDK4, Hsc70, Tsg101, and actin of EVs from RA-induced N2A cells for 2, 4, 6, and 8 d (D2, D4, D6, and D8). (B) Immunoblots of pRB, p57, p27, p21, pErk, and actin in differentiated PC12 cells and EVs. (C) Immunoblots of cyclin D1, CDK4, and multiple EV markers from the N6-EVs treated with different concentrations of proteinase K (PK), with or without 1% Triton X-100.

Article Snippet: The cyclin D1–APEX plasmids were constructed by combining the PCR fragment of cyclin D1 from the cyclin D1–Flag plasmid and APEX from pcDNA3 APEX-nes (49386; Addgene) into XPack CMV constructs (System Biosciences).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: The chaperone protein Hsc70 facilities cyclin D1 package into EVs. (A) Characterization of APEX-mediated proximity biotinylation of cyclin D1 protein targets by blotting with streptavidin. Cyclin D1–APEX fusion gene was delivered into N2A cells by lentivirus infection. Biotinylated protein was detected by blotting with streptavidin (SA)-HRP. Ponceau S staining (left) of the same membrane served as loading control. (B) Table showing MS analysis of the unique peptides in biotin-phenol together with H 2 O 2 (B+H) or without H 2 O 2 (B). (C) CoIP analysis of Hsc70 and Hsc90 with cyclin D1 and CDK4 in N2A cells. (D) CoIP of cyclin D1 and Hsc70 in PC12 cells. (E) CoIP of cyclin D1 and Hsc70 in 5 × 10 10 RA-EVs. (F) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells treated with VER-155008 (VER). N2A cells pretreated with RA-containing differentiation medium for 4 d, after which cells were exposed to fresh differentiation medium with or without 5 µM VER for two more days. EVs collected from 6-d differentiation of N2A cells. (G) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells transfected with WT Hsc70 (WT) or D10N mutant Hsc70 (D10N; >50% transfection efficiency). WT Hsc70 or D10N mutant Hsc70 were transfected by Lipofectamine 2000 in seven plates of 70%-confluency N2A cells in DMEM medium for 10 h, followed by a change to fresh differentiation medium for 3 d. EVs were collected from both cells. (H) Expression analysis of Pax6 , Nestin , and Six3 in differentiated mESCs treated with 2 × 10 9 EVs from RA-induced N2A cells with (VER-EV) or without (RA-EV) VER. EVs were collected as described in F. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (I) Immunoblots of Hsc70 and actin in control or Hsc70 sgRNA–transfected N2A cells. dCas9 was stably expressed in N2A cells by lentivirus (dCas9), Lentivirus was then used to introduce Hsc70 sgRNA1/2 by transfection of dCas9 cells. (J) Expression analysis of Pax6 and Nestin in differentiated mESCs treated with 2 × 10 9 EVs from RA-induced N2A dCas9 cells or Hsc70 sgRNA–transfected cells. Values represent the mean ± SD, from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples.

Article Snippet: The cyclin D1–APEX plasmids were constructed by combining the PCR fragment of cyclin D1 from the cyclin D1–Flag plasmid and APEX from pcDNA3 APEX-nes (49386; Addgene) into XPack CMV constructs (System Biosciences).

Techniques: Infection, Staining, Western Blot, Transfection, Mutagenesis, Expressing, Stable Transfection, Introduce

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: Cyclin D1 is important for EV-mediated neural induction of mESCs. (A) Immunostaining of GFP (green, Alexa fluor 488) and CD9 (red, Alexa fluor 568) in differentiated mESC cells without (control) or with cyclin D1–GFP EV treatment. Magnified view is shown in panel 3. Nuclei were stained with DAPI. Scale bars, 5 µm. (B) Immunoblots of cyclin D1, actin, and GFP of differentiated mESCs without incubation or incubated for 4 d with cyclin D1–GFP EVs. Quantification of fusion protein uptake was calculated as the ratio of exogenous cyclin D1–GFP to endogenous cyclin D1. (C) Schematic of biotinylation labeling of cyclin D1–APEX EVs. (D) Streptavidin-HRP blotting analysis of biotinylated proteins in cyclin D1–APEX-expressing EVs. EVs were treated with biotin-phenol together with H 2 O 2 (B+H) or not (B). Biotinylated protein was detected by blotting with streptavidin (SA)-HRP. Ponceau S staining (left of panel) of the same membrane serves as loading control. (E) Schematic of mESCs treated with cyclin D1–APEX EVs and biotinylated proteins labeled in differentiated mESCs. (F) SA-HRP blotting of biotinylated proteins in mESCs treated with cyclin D1–APEX EVs. (G) Venn diagram of the MS results. MS sample was collected as described in Materials and methods. Immunoprecipitation with streptavidin was used to enrich the biotinylated proteins. Diagram generated by Venn diagram package in the R program for statistical computing. (H) GO analysis of the MS results shown in G. GO analysis was generated by topGO package in the R program for statistical computing. (I) After the treatment described in E and F, immunoblots of Lin28 and nucleolin in differentiated mESCs treated with cyclin D1–APEX EVs. (J) Cyclin D1 was increased in the EVs from N2A cells overexpressing cyclin D1 (OE). The protein level of cyclin D1 was detected in control and OE samples. Actin was used as the internal control of whole-cell lysate, and Tsg101 was used as the loading control of EVs. (K) Gene expression level of Pax6 , Six3 , and Map2 was determined in differentiated mESCs treated without EVs and with RA-EV or OE EVs. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; **, P < 0.01). Error bars represent SD from independent samples. (L) Quantitative analysis of the percentage of cells containing Pax6 normalized to DAPI stain in differentiated mESCs treated without EVs and with RA-EV or OE EVs. The values represent the mean ± SD, from two independent experiments (*, P < 0.05). Error bars represent SD from independent samples. (M) Cyclin D1 was absent from cyclin D1 knockout N2A cells and the EVs from cyclin D1 knockout (KO) N2A cells. The cyclin D1 protein was detected in control and KO samples. (N) The expression of Pax6 , Six3 , and Map2 was analyzed in differentiated mESCs treated without EVs and with RA-EV or cyclin D1 KO EVs. The values represent the mean ± SD from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (P) Quantitative analysis of the percentage of cells containing Pax6 normalized to DAPI stain in differentiated mESCs treated without EVs and with RA-EV or cyclin D1 KO EVs. The values represent the mean ± SD, from two independent experiments (*, P < 0.05). Error bars represent SD from independent samples.

Article Snippet: The cyclin D1–APEX plasmids were constructed by combining the PCR fragment of cyclin D1 from the cyclin D1–Flag plasmid and APEX from pcDNA3 APEX-nes (49386; Addgene) into XPack CMV constructs (System Biosciences).

Techniques: Immunostaining, Staining, Western Blot, Incubation, Labeling, Expressing, Immunoprecipitation, Generated, Knock-Out

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: MS analysis of RA-EV and cyclin D1–KO EV. (A) Whole-protein profile of RA-EV and cyclin D1–KO EV was analyzed by MS. The proteome (1,339 proteins) overlapped extensively in these two preparations. The list of the EV proteins shown in .

Article Snippet: The cyclin D1–APEX plasmids were constructed by combining the PCR fragment of cyclin D1 from the cyclin D1–Flag plasmid and APEX from pcDNA3 APEX-nes (49386; Addgene) into XPack CMV constructs (System Biosciences).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

doi: 10.1083/jcb.202101075

Figure Lengend Snippet: Model. Neural development includes early-stage neural induction and late-stage neural genesis. During neural genesis, PC12 or N2A cells (dark green) respond to NGF or RA to differentiate into neuronal cells (bright green). The content of EVs exhibits dynamic changes corresponding to the fate conversion. Cyclin D1 (magenta dots inside the purple EVs) was enriched in EVs from differentiating neurons. Additional cyclin D1 enriched in EVs from the neuronal cells accelerates the commitment of mESCs (light orange) to neural progenitor cells (mNPC, light green). Exosomal communication between different development stages may contribute to commitment and conversion of mESCs to the neural lineage.

Article Snippet: The cyclin D1–APEX plasmids were constructed by combining the PCR fragment of cyclin D1 from the cyclin D1–Flag plasmid and APEX from pcDNA3 APEX-nes (49386; Addgene) into XPack CMV constructs (System Biosciences).

Techniques:

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Tuning VLP viral tropism by altering viral glycoprotein. ( A ) Different types of VLPs were produced using the 3P system by varying the viral glycoprotein (VSV-G, SARS2 spike, SARS spike, or MERS spike) and reporter genes (luciferase or EGFP). ( B ) One microgram of the viral glycoprotein was used to create various 3P Luc-PS9 VLPs and these were used to infect five cell types: WT 293T (293T), 293T-hACE2, A549-hACE2-TMPRSS2, Calu-3, and 293T-DPP4. SARS2 and SARS spike VLPs displayed similar tropism and entered only hACE2-expressing cells (293T-hACE2, A549-hACE2-TMPRSS2, and Calu-3), with SARS exhibiting higher luminescence intensity compared with SARS2. MERS VLPs infected Calu-3 cell at low level and efficiently entered 293T-DPP4 cells. VSV-G VLPs entered all cell types. ( C ) 3P EGFP-PS9 VLPs were produced with 4 μg VSV-G, 1 μg SARS2 spike, or 1 μg MERS spike plasmid. VSV-G VLPs entered all cell types. SARS2 VLPs only entered ACE2 cells. MERS VLPs only entered DPP4 cells. Methods provide detailed steps for VLP production. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: The pCDNA3.3 Middle East respiratory syndrome (MERS) D12 S plasmid encoding the MERS wild-type (WT) S protein with a 12-amino acid deletion on the C-terminal tail was from David Nemazee ( RRID: Addgene_170448). pcDNA3.1 SARS S plasmid encoding the 2002 SARS S protein was kindly provided by Fang Li ( RRID: Addgene_145031).

Techniques: Produced, Luciferase, Expressing, Infection, Plasmid Preparation