pcdna3 myc cyclin d1  (Addgene inc)

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: 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: 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, Control, 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, Membrane, Control, 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, Control, Staining, Western Blot, Incubation, Labeling, Expressing, Membrane, Immunoprecipitation, Generated, Gene Expression, 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: