Journal: Cell chemical biology

Article Title: Discovery of covalent CDK14 inhibitors with pan-TAIRE family specificity

doi: 10.1016/j.chembiol.2019.02.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CDK3 / Cyclin A , Signal Chem , Cat # C30-10G-10.

Techniques: Recombinant, Kinase Assay, Sequencing, Software

Journal: PLoS ONE

Article Title: Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

doi: 10.1371/journal.pone.0149739

Figure Lengend Snippet: a. Kinase activity of LRRK2 mutants tested by kinase assay using MBP as substrate. HEK293 cells were transfected with HA-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N as indicated. After 24 h, LRRK2 was immunoprecipitated in the lysates by anti-HA antibody, and in vitro LRRK2 kinase assay was performed to measure the LRRK2 kinas activity using purified MBP protein as substrate. The bottom panel shows equal expression of HA-LRRK2 and MBP used in the lysates. b. The graph is the quantification of LRRK2 kinase activities in Fig 1A. The numbers are relative values, with WT set to 1. c. Kinase activity of LRRK2 mutants tested by kinase assay using LRRKtide as substrate. HA-tagged WT LRRK2 or mutants were immunoprecipitated as above, then in vitro LRRK2 kinase assay was performed using LRRKtide as substrate. d. Overexpression of R1628P mutation do not cause neuronal cell death. Primary-cultured cortical neurons were transfected with GFP-tagged WT LRRK2 or mutants in the Roc-COR-Kinase domain, including R1441C, R1628P, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N. After 48 h transfection, the dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. All the above results represent at least three independent experiments as the mean ± SD, *P<0.05, **P<0.01 (ANOVA).

Article Snippet: GST-tagged LRRK2-COR (COR domain, amino acids 1535~1878) was obtained by PCR using FL-HA-LRRK2-WT as template and subcloned to pGEX-2T1 vector (Addgene).

Techniques: Activity Assay, Kinase Assay, Transfection, Mutagenesis, Immunoprecipitation, In Vitro, Purification, Expressing, Over Expression, Cell Culture, Labeling

Journal: PLoS ONE

Article Title: Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

doi: 10.1371/journal.pone.0149739

Figure Lengend Snippet: a. The R1628P mutation increase the binding affinity of LRRK2 with Cdk5. Primary-cultured cortical neurons were transfected with vehicle or LRRK2 (WT, R1628P). After 24 h, the exogenous LRRK2 was immunoprecipitated using anti-HA antibody, and the level of bound Cdk5 was measured by Western blotting. The bottom panel shows the loading control of HA-LRRK2 and Cdk5 used in the lysates. b. Cdk5 phosphorylates Serine 1627 (S1627) in R1628P mutant. Recombinant GST-tagged LRRK2 (COR domain, amino acids 1535~1878), including wild-type (WT), R1628P mutant and S1627A:R1628P double mutant, were purified. The GST-tagged LRRK2 recombinant protein were phosphorylated by active Cdk5/p35 and γ- 32 P-ATP in vitro , and the phosphorylation signals were analyzed by autoradiography. (top panel, about 60KD). The same membrane was probed with anti‑GST, Cdk5 or p35 antibody as a loading control (bottom panel). c. Cdk5 phosphorylates S1627 in cells. The HA-tagged LRRK2 (WT, R1628P and S1627A:R1628P) plasmids were cotransfected with Cdk5 and p35 in HEK293 cells. After 24 h of transfection, the LRRK2 were immuneprecipitated using an anti-HA antibody from lysates, and phosphorylation of LRRK2 were measured by Western blotting using a phospho-(Serine/Threonine)-Proline (pS/TP) antibody and anti-LRRK2 phospho S935 antibody. HA-LRRK2, Cdk5, p35, and actin levels were determined by Western blotting as a loading control.

Article Snippet: GST-tagged LRRK2-COR (COR domain, amino acids 1535~1878) was obtained by PCR using FL-HA-LRRK2-WT as template and subcloned to pGEX-2T1 vector (Addgene).

Techniques: Mutagenesis, Binding Assay, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Recombinant, Purification, In Vitro, Autoradiography

Journal: PLoS ONE

Article Title: Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

doi: 10.1371/journal.pone.0149739

Figure Lengend Snippet: a. Plasmids of HA-tagged wild-type (WT) or R1628P mutant LRRK2 were cotransfected with Cdk5/p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and in vitro LRRK2 kinase assay was performed to measure the LRRK2 kinas activity using purified MBP protein as substrate. HA-LRRK2, Cdk5, and p35 levels were determined by Western blotting as a loading control. b. The graph is the quantification of LRRK2 kinase activities in Fig 3A. The numbers are relative values, with WT w/o Cdk5/p35 set to 1. c. Plasmids of HA-tagged wild-type (WT), R1628P, and S1627A:R1628P mutant LRRK2 were cotransfected with Cdk5 or dominant-negative form of Cdk5 (dnCdk5) and p35 in HEK293 cells, as indicated. After 24 h, LRRK2 was immune precipitated in the lysates by anti-HA antibody, and in vitro LRRK2 kinase activity was measured for 30min using LRRKtide as substrate. The results represent as the mean ± SD, **P<0.01 (compared with WT:control group) ##P<0.01 (compared with WT:Cdk5/p35 group) (ANOVA). d. Phosphorylation mimic of S1627 (S1627D) increased the LRRK2 kinase activity. HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P double mutant. LRRK2 kinase activities were measured as above. e. The graph is the quantification of LRRK2 kinase activities in Fig 3D. The numbers are relative values, with WT set to 1. **P<0.01 (ANOVA) f. HEK293 cells were transfected with HA-tagged LRRK2 plasmids, including wild-type (WT), R1628P, S1627D and S1627D:R1628P. LRRK2 kinase activities were measured as above for indicated period of time using LRRKtide as substrate. **P<0.01 (compared with WT group, ANOVA)

Article Snippet: GST-tagged LRRK2-COR (COR domain, amino acids 1535~1878) was obtained by PCR using FL-HA-LRRK2-WT as template and subcloned to pGEX-2T1 vector (Addgene).

Techniques: Mutagenesis, In Vitro, Kinase Assay, Activity Assay, Purification, Western Blot, Dominant Negative Mutation, Transfection

Journal: PLoS ONE

Article Title: Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

doi: 10.1371/journal.pone.0149739

Figure Lengend Snippet: a. Neurons with the R1628P mutation display a higher sensitivity to MPP+. Primary-cultured cortical neurons from wild-type mice were transfected with GFP-tagged wild-type (WT) or R1628P mutant LRRK2 plasmids for 24 h, and then exposed to MPP+ (30 μM) for 24 h. The dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. b. Cdk5 deletion protects the neurons with the R1628P mutation from MPP+ toxicity. The above procedures were performed in primary-cultured cortical neurons from the neuronal Cdk5 conditional knockout mice. Single cell survival assay was conducted as above. The results represent at least three independent experiments as the mean ± SD, **P<0.01 (ANOVA). c. The higher sensitivity of R1628P to MPP+ requires the phosphorylation of S1627 on LRRK2. Primary-cultured cortical neurons from wild-type mice were transfected with GFP vector, GFP-tagged wild-type (WT) LRRK2 or R1628P, S1627A:R1628P, S1627D:R1628P mutant for 24 h, and then exposed to MPP+ (30 μM) for 24 h. The dead cells were labeled with EthD-1 in red, and 200 GFP-positive neurons were counted to calculate the percentage of cell death. The results represent at least three independent experiments as the mean ± SD, *P<0.05, (ANOVA)

Article Snippet: GST-tagged LRRK2-COR (COR domain, amino acids 1535~1878) was obtained by PCR using FL-HA-LRRK2-WT as template and subcloned to pGEX-2T1 vector (Addgene).

Techniques: Mutagenesis, Cell Culture, Transfection, Labeling, Knock-Out, Clonogenic Cell Survival Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity

doi: 10.1371/journal.pone.0149739

Figure Lengend Snippet: The Schematic diagram shows that the R1628P genetic mutation of LRRK2 provide a potential “two-hit” target of environment toxic MPP+-induced Cdk5 activation, and Cdk5 could phosphorylate the adjacent amino residue S1627 of R1628P mutation, thus activate LRRK2 kinase activity and cause neuronal death.

Article Snippet: GST-tagged LRRK2-COR (COR domain, amino acids 1535~1878) was obtained by PCR using FL-HA-LRRK2-WT as template and subcloned to pGEX-2T1 vector (Addgene).

Techniques: Mutagenesis, Activation Assay, Activity Assay

Journal: Cancer medicine

Article Title: Identification of an EMT-related gene-based prognostic signature in osteosarcoma.

doi: 10.1002/cam4.5942

Figure Lengend Snippet: FIGURE 8 In vitro validation on CDK3. Representative immunohistochemical images of expressions of CDK3 in para-carcinoma (A) and OS (B) tissues. (C) The relative expression level of CDK3 in normal control, siNC, and siCDK3 groups in U2OS and MNNG/HOS. (D) EdU-positive ratios were calculated (n = 3) in normal control, siNC, and siCDK3 groups in U2OS and MNNG/HOS. (E) Migration cell numbers (n = 3) were counted after si- RNA transfection. Representative images of EdU (red), Hoechst staining (blue), and transwell (purple) results in normal control, siNC, and siCDK3 groups in U2OS (F) and MNNG/HOS (G). Ordinary one-way ANOVA test ***p < 0.001; ****p < 0.0001; ns, no significance.

Article Snippet: Samples were incubated with primary antibodies against CDK3 (Proteintech) overnight at 4°C, followed by reaction enhancer for 20 min at 37°C, and enhanced enzymeconjugated sheep anti- mouse/rabbit IgG polymer for 20 min at 37°C.

Techniques: In Vitro, Biomarker Discovery, Immunohistochemical staining, Expressing, Control, Migration, Transfection, Staining

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: Schematic illustration of the SILAC-based quantitative phosphoproteomic approach. (A) Representative Western blot of Ptp1b +/+ and Ptp1b −/− cells. (B) Ptp1b +/+ and Ptp1b −/− cells were cultured in “light” (red) or “heavy” (blue) medium and stimulated with EGF for 15 min. After lysis, the samples were mixed and incubated with antiphosphotyrosine antibodies for enrichment of tyrosine-phosphorylated proteins. Then, the phosphoprotein fraction was digested with trypsin, and phosphopeptides were enriched again with TiO 2 beads. Phosphopeptides were analyzed by LC-MS/MS. (C) Proteins identified in this assay were classified according to their cellular function.

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Multiplex sample analysis, Western Blot, Cell Culture, Lysis, Incubation, Liquid Chromatography with Mass Spectroscopy, Cell Function Assay

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: Predicted structure of PTP1B in complex with some of the putative substrates identified by SILAC-based phosphoproteomics. (A) Visualization of the complex of Cdk3 (yellow) and the catalytic domain of PTP1B (grey). The catalytic residue Cys215 of PTP1B and pTyr15 of Cdk are indicated in green and red, respectively. (B) Closer view of the PTP1B-Cdk3 interaction. The distance between pTyr15 of Cdk3 and Cys215 of PTP1B is indicated. (C) Visualization of the complex of IR β (yellow) and PTP1B (grey). The catalytic residue Cys215 indicated in green is close to IR β pTyr1146 indicated in red. (D) Closer view of the PTP1B-IR β interaction. The distance between Cdk3 pTyr1146 and PTP1B Cys215 is indicated. (E) Visualization of the complex of DOK1 (yellow) and PTP1B (grey). The catalytic residue Cys215 indicated in green is close to DOK1 pTyr362 indicated in red. (F) Closer view of the PTP1B-DOK1 interaction. The distance between DOK1 pTyr362 and PTP1B Cys215 is indicated.(G) Visualization of the complex of EphA (yellow) and PTP1B (gray). The catalytic residue Cys215 indicated in green is close to EphA pTyr205 indicated in red. (F) Closer view of the PTP1B-EphA interaction. The distance between EphA pTyr205 and PTP1B Cys215 is indicated.

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Multiplex sample analysis, Phospho-proteomics, Residue

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: Minimal distance from the catalytic residues on PTP1B’s phosphatase domain to the phosphorous atom of its substrates

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Ligand Binding Assay

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: PTP1B dephosphorylates a Cdk3 derived peptide in vitro and both proteins interact in GB cells. (A) The in vitro activity of PTP1B against a phosphopeptide corresponding to residues 9-21 of Cdk3 (KIGEGT pY GVVYKA) was determined by using a malachite-green based assay. An IR β phosphopeptide (TRDI pY ETDYYRK) was used as positive control and the nonphosphorylated peptides of Cdk3 and IR β were used as negative controls. Each assay was independently repeated with four replicates and the nmol of phosphate released were calculated. Data are presented as means ± SE. (B). Assessment of Cdk3 activity in Ptp1b +/+ and Ptp1b −/− MEFs. Cell lysates were subjected to Western blot with anti-PTP1B, anti-Cdk3 or anti-phospho Cdk3 antibodies. GAPDH was used as loading control. (C) Co-immunoprecipitation of ectopically expressed wild-type or trapping mutant PTP1B and Cdk3. Cell lysates of transfected cells incubated with or without 1 mM of sodium orthovanadate were subjected to immunoprecipitation with anti-myc or isotype control IgG antibodies and the presence of Cdk3 in the complex was assessed with anti-HA antibodies. The presence of PTP1B and Cdk3 in cell extracts prior to immunoprecipitation was verified using specific antibodies (input). (D) Co-immunoprecipitation of endogenous PTP1B and Cdk3. LN229, U87-MG and U251 cell lysates were subjected to immunoprecipitation with anti-PTP1B or isotype control IgG antibodies and the presence of Cdk3 in the complex was assessed with anti-Cdk3 antibodies. The presence of PTP1B and Cdk3 in cell extracts prior to immunoprecipitation was verified using specific antibodies (input). (E) The physical interaction between endogenously expressed PTP1B and Cdk3 in LN229, U87-MG and U251 GB cells was determined using proximity ligation assays. The figure shows representative confocal microscopy images in which PTP1B-Cdk3 interactions appear as individual fluorescent red dots (lower panels). Anti-mouse and anti-rabbit isotype IgG antibodies were used as negative controls (upper panels).

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Derivative Assay, In Vitro, Activity Assay, Phospho-proteomics, Malachite Green Assay, Positive Control, Western Blot, Control, Immunoprecipitation, Mutagenesis, Transfection, Incubation, Ligation, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: PTP1B and Cdk3 depletion impairs cell proliferation. (A) GB cells were transfected with siRNAs targeting PTP1B or Cdk3, and the expression of both proteins was assessed by immunoblot. GAPDH was used as loading control. (B) GB cells were synchronized at G0 by serum deprivation and incubated 3 h with vehicle, claramine 2 µM, trodusquemine 2 µM, or transfected with nontargeting siRNAs or siRNAs targeting PTP1B or Cdk3. Cells were counted every 24 h. Proliferation of LN229, U87-MG and U251 cells is represented as mean ± SE of three independent experiments. (C) Ptp1b +/+ and Ptp1b −/− MEFs were synchronized at G0 by serum deprivation and incubated 3 h with vehicle, claramine 2 µM, trodusquemine 2 µM. Cells were counted every 24 h. Proliferation of Ptp1b +/+ and Ptp1b −/− MEFs is represented as mean ± SE of three independent experiments.

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

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

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: PTP1B reduced activity impairs Cdk3 activation and induces cell cycle arrest in human GB cells. GB cells were synchronized at G0 by serum deprivation for 48 h and incubated 3 h with vehicle (A), claramine 2 µM (B), or trodusquemine 2 µM. Cell cycle arrest was released by the addition of 10% FBS and cells were collected at the indicated times. The activity of Cdk3 was assessed by immunoblot. GAPDH was used as loading control. (D) GB cells were synchronized at G0 and incubated 3 h with vehicle, claramine 2 µM or trodusquemine 2 µM as previously mentioned, or transfected with nontargeting siRNAs or siRNAs targeting PTP1B or Cdk3. Cell cycle arrest was released by the addition of 10% FBS, cells were fixed at indicated time points and stained with propidium iodide. Quantification of the percentage of cells at each phase is represented. Statistical differences between control and experimental groups of cells are indicated (* P < 0.05). Data are representative of three independent experiments.

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Activity Assay, Activation Assay, Incubation, Western Blot, Control, Transfection, Staining

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: PTP1B reduced activity negatively affects Cdk3/Rb/E2F signaling in human GB cells. (A) The activities of Cdk3 and Rb in GB cells treated with vehicle, claramine 2 µM or trodusquemine 2 µM were assessed by immunoblot using total and phospho-specific antibodies. (B) The activities of Cdk3 and Rb in GB cells transfected with siRNAs targeting PTP1B were assessed by immunoblot using total and phospho-specific antibodies. (C) The expression levels of the E2F target genes: Cdk1, Cyclin A, and Cyclin E1 were assessed by RT-qPCR. All data were normalized to control GAPDH. Fold changes were calculated using the ΔCt method (2 -ΔΔCt ). Data are representative of three independent experiments.

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Activity Assay, Western Blot, Transfection, Expressing, Quantitative RT-PCR, Control

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: Activated Cdk3 bypasses requirement for PTP1B for proliferation and cell cycle progression. (A) GB cells were transfected with a plasmid encoding a constitutively active Cdk3 mutant. The presence of Cdk3 AF in cell extracts was verified by Western blot using anti-HA antibodies. (B) GB cells were mock transfected or transfected with a plasmid encoding a constitutively active Cdk3 mutant and incubated 3 h with vehicle, claramine 2 µM or trodusquemine 2 µM. Cells were counted every 24 h. Proliferation of LN229, U87-MG and U251 cells is represented as mean ± SE of three independent experiments. (C) GB cells were mock transfected or transfected with a plasmid encoding a constitutively active Cdk3 mutant, synchronized at G0 and incubated 3 h with vehicle, claramine 2 µM or trodusquemine 2 µM as previously described. Cell cycle arrest was released by the addition of 10% FBS, cells were fixed at indicated time points and stained with propidium iodide. Quantification of the percentage of cells at each phase is represented in the bar graphics. Statistical differences between control and experimental groups of cells are indicated (* P < 0.05). Data are representative of three independent experiments. (D) GB cells were mock transfected or transfected with a plasmid encoding a constitutively active Cdk3 mutant, synchronized at G0 and incubated 3 h with vehicle, claramine 2 µM or trodusquemine 2 µM as previously mentioned. Cell cycle arrest was released by the addition of 10% FBS. The expression levels of the E2F target genes: Cdk1, cyclin A, and cyclin E1 were assessed by RT-qPCR. All data were normalized to control GAPDH. Fold changes were calculated using the ΔCt method (2 -ΔΔCt ). Data are representative of three independent experiments.

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Incubation, Staining, Control, Expressing, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway

doi: 10.1080/10985549.2023.2273193

Figure Lengend Snippet: Proposed model for PTP1B regulation of cell cycle. PTP1B dephosphorylates and activates Cdk3, which in turn phosphorylates Rb, favoring its dissociation from E2F and promoting the expression of genes needed for the progression to S phase.

Article Snippet: The primer sequences for mutagenesis were Cdk3 AF Fwd: 5′ aagatcggagagggc g cct t tggggtggtgtacaa 3′ and Cdk3 AF Rev: 5′ ttgtacaccacccca a agg c gccctctccgatctt 3′. siRNAs targeting human PTP1B (sc-36328) and Cdk3 (sc-37578) were obtained from Santa Cruz (Dallas, TX, USA).

Techniques: Expressing

Journal: Immunity

Article Title: Sympathetic neuronal activation triggers myeloid progenitor proliferation and differentiation

doi: 10.1016/j.immuni.2018.05.004

Figure Lengend Snippet: Key Resources Table

Article Snippet: Mouse Cdk3 , Taqman , Mm00518387_m1.

Techniques: cDNA Synthesis, Selection, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Red Blood Cell Lysis, Recombinant, SYBR Green Assay, Plasmid Preparation, Flow Cytometry, Immunofluorescence, Western Blot, Software

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Photostimulation was applied at 470 nm (4.0 mW/cm 2 ). Data were shown as mean ± sem. Scale bar, 5 µm. a Domain architecture of the human STIM1. SP, signal peptide; EF-SAM, EF-hand and sterile alpha-motif; TM, transmembrane domain; CC1, coiled-coil domain 1; SOAR, STIM-Orai activating region; P/S, proline/serine-rich region; TRIP, the S/TxIP microtubule-binding motif; PB, polybasic tail. b Schematic of STIM1–ORAI1 coupling at the ER–PM junction that mediates store-operated Ca 2+ entry. c – e Use of the iLID-sspB optical dimerizer to trigger STIM1ct activation and Ca 2+ influx through endogenous ORAI channels. c Schematic of the design. iLID or sspB was fused to the N-terminus of STIM1ct at residue 233. d Confocal images showing photoswitchable Ca 2+ influx in HeLa cells co-transfected with a red Ca 2+ sensor (R-GECO 1.2) and the iLID/sspB fused STIM1ct chimeras. Cells were exposed to two repeated dark-light cycles. e Quantitative analysis of Ca 2+ signals in response to repeated photostimulation ( n = 40 cells from three independent experiments). The half-lives ( t 1/2 ) of on and off kinetics were fitted with one phase exponential decay (“±” means 95% confidence interval). f – h Use of the CRY2-CIBN optical dimerizer to photo-activate STIM1ct and Ca 2+ influx. f Schematic of the design. CRY2 was used to photo-crosslink CIBN-STIM1ct and trigger STIM1ct activation to induce Ca 2+ entry. g Confocal images showing light-induced co-localization of mCherry (mCh)-tagged CIBN-STIM1ct with YFP-ORAI1 in HeLa cells. h Reversible Ca 2+ responses monitored by R-GECO 1.2 ( n = 30 cells). Blue bar, photostimulation at 470 nm with a power density of 4 mW/cm 2 . i – k ER-tethered CRY2-STIM1ct mimics STIM1 puncta formation at ER–PM junctions to evoke localized Ca 2+ influx. i Schematic of the design. j Confocal images illustrating light-induced clustering of ER-resident CRY2-STIM1ct at the footprint of HeLa cells. Enlarged views of the boxed regions were shown on the right. k Cytosolic Ca 2+ signals reported by R-GECO1.2 in HeLa cells subjected to two repeated dark-light cycles ( n = 30).

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Binding Assay, Activation Assay, Transfection

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of a split STIM1 molecule (at residue 342) to monitor CC1–SOAR interaction in trans at real time. CC1–SOAR maintains STIM1ct in an inactive configuration at rest. As a result, Part II (mCh-STIM1 342–685 ) tightly docks to the ER-resident Part I (STIM1 1–342 -YFP) when the store remains full. Upon store depletion, structural changes propagate toward the CC1 region to weaken its association with SOAR, thereby leading to the cytosolic dispersion of Part II as shown in panel b . b Confocal images showing the distribution of split STIM1 molecules (green, STIM1 1–342 -YFP; red, mCh-STIM1 343–685 ) before and after thapsigargin (TG)-induced store depletion in HeLa cells. c Schematic illustrating the design of a LOV2-SOAR (STIM1 336–486 ) chimera to mimic the CC1–SOAR interaction that locks STIM1ct in an inactive state. CC1 is replaced by LOV2 (light-oxygen-voltage domain 2) to tightly cage SOAR in the dark. Upon blue light stimulation, the Jα helix becomes disordered to uncage SOAR, thereby restoring its activity to engage and gate ORAI channels. If the ER-resident Part I (STIM1 1–342 -YFP) and PM-embedded ORAI1 are co-expressed, LOV2-SOAR can be used to determine the relative binding strength of SOAR toward ER-anchored CC1 or PM-resident ORAI1 channels. d Light-inducible cytosol-to-ER translocation of mCh-LOV2-SOAR in HEK293 cells co-transfected with Part I as shown in panel c . e Quantification of cytosolic mCherry signals (images in panel d ) following three repeated light-dark cycles ( n = 18 cells). f – h Comparison of the relative strength of SOAR-CC1 and SOAR-ORAI1 interactions. f Top: Light-induced cytosol-to-PM translocation of mCh-LOV2-SOAR (gray) observed in HEK293 cells co-transfected with YFP-ORAI1 (green). Bottom: Confocal images of HEK293 cells co-expressing mCh-LOV2-SOAR (gray), YFP-ORAI (green) and STIM1 1–342 -CFP (cyan). In the dark, mCh-LOV2-SOAR was evenly distributed in the cytosol. Upon photostimulation, mCh-LOV2-SOAR preferred to translocate toward ER membrane but not to PM. g The fluorescence intensities (YFP, green; mCh, red; CFP, cyan) across the dashed line were plotted to evaluate the degree of colocalization. h Light-induced Ca 2+ response curves (quantified by GCaMP6s) in HEK293 cells transfected with LOV2-SOAR (red), LOV2-SOAR + ORAI1 (green) or LOV2-SOAR + Part I (STIM1 1–342 ; blue). n = 30 cells.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Activity Assay, Binding Assay, Translocation Assay, Transfection, Expressing, Fluorescence

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of an optogenetic clustering assay to examine real-time protein–protein interactions in living cells. b Summary of mCh-tagged baits and YFP-tagged preys used to map critical domains in STIM1ct that dictate STIM1 oligomerization. c Representative confocal images showing the intracellular distribution of the bait (mCh-CRY2-STIM1 233–448 ) and two different preys (P1—top panel, YFP-STIM1 233–658 ; P3—bottom panel, YFP-STIM1 233–342 ) before and after blue light stimulation in HeLa cells. d Time courses showing the kinetics of light-induced clustering (F cluster /F neighbor ) of the bait and its co-clustering with P1 (blue), but not with P3 (green), as seen in panel c . n = 18 cells. ( e ) Quantification of the degrees of light-inducible co-clustering for the five indicated preys in HeLa cells co-transfected with the bait. n = 18 cells from three independent experiments. f Summary of the optogenetic co-clustering assay results for the bait–prey combinations shown in panel b . “+” means co-clustering notably observed after photo-illumination; “−” means no appreciable cluster formation before and after blue light stimulation. g – i Light inducible co-clustering to dissect the STIM1 luminal EF-SAM domain. g Schematic showing the design of bait–prey constructs. h – i Representative confocal images of HeLa cells co-expressing ( h ) mCh-CRY2-EF (B4, STIM1 32–128, EF-hand ; red) with YFP-EF (P6, green), or ( i ) mCh-CRY2-SAM (B5, STIM1 128–200, SAM ; red) with YFP-SAM (P7; red) under dark (left) and blue light (right). The selected regions (dashed boxes) were enlarged to aid visualization. Scale bar 5 µm.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Transfection, Construct, Expressing

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Design of the high-throughput screening pipeline. The cytosol-to-PM translocation and Ca 2+ influx (GCaMP6s as readout) were used as two readouts. b Sequence alignment of human SOAR1 and SOAR2 domains and the 3D structure of SOAR1 (PDB entry: 3TEQ). Key residues at the interdimer interface or involved in ORAI1-binding were indicated by dots and triangles, respectively. Selected key residues were highlighted in the 3D structure. c Quantification of Ca 2+ responses (GCaMP6s) and PM translocation (mCherry signals) of selected CRY2-STIM1ct mutants before (dark dots) and after (blue dots) photostimulation. HeLa-GCaMP6s stable cells were co-transfected with each of the indicated mCh-CRY2-STIM1ct mutants and ORAI1-CFP. d Time courses showing the kinetics of light-induced Ca 2+ influx for WT and the indicated mCh-CRT2-STIM1ct variants. n = 60 cells. e – g Representative confocal images ( e ) and quantification of intracellular Ca 2+ signals, n = 60 cells. Box-whisker plots indicated the median, and the interquartile range with 5–95 percentile distribution. f , as well light-induced PM translocation, n = 8 cells ( g ), in HeLa cells expressing WT or the indicated mCh-CRY2-STIM1ct mutants. h Representative confocal images of HEK293 S1-KO cells expressing the GFP-tagged full-length STIM1-T393F mutant before and after TG-induced store depletion. i SOCE monitored by R-GECO1.2 in HEK293 S1-KO cells expressing GFP-STIM1 WT or the mutant T393F. n = 90 cells. j Summary of the degrees of Ca 2+ influx and PM translocation of cancer-associated mutations found in the SOAR domains of STIM1. HeLa cells were transfected with the indicated mCh-CRT2-STIM1ct mutants. Gain-of-function (H395Y and R424W; red) and loss-of-function (L402R, R426L/C, R429C; green) mutations were both identified. n = 60 cells.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: High Throughput Screening Assay, Translocation Assay, Sequencing, Binding Assay, Transfection, Whisker Assay, Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

doi: 10.1038/s41467-020-14841-9

Figure Lengend Snippet: Data were shown as mean ± sem. Scale bar, 5 µm. a Diagram of the STIM1 C-terminal fragment (residues 443–685) that contains both the S/TxIP EB1-binding motif and the positively charged polybasic tail (PB) that interacts with PM-resident PIPs. Mutations used in this study to perturb STIM1ct-target interactions were highlighted in red. b , c Schematic ( b ) and representative confocal images ( c ) showing light-inducible bimodal distribution (tracking of MT plus ends or cytosol-to-PM translocation) of mCh-CRY2-STIM1 443–685 in COS-7 cells. d Time courses of light-triggered MT plus-end tracking (green) and PM translocation (blue) of mCh-CRY2-STIM1 443–685 . Upon blue light illumination, cytosolic mCherry signals rapidly reduced ( t 1/2 = 18.2 ± 5.0 s), accompanied with the increase of MT tip tracking ( t 1/2 = 21.5 ± 7.3 s) or PM decoration ( t 1/2 = 27.6 ± 6.2 s). n = 12 cells. e , f Representative confocal images ( e ) and quantifications of MT plus-end tracking ( f ) of mCh-CRY2-STIM1ct variants (top, WT; bottom, P645N in the context of STIM1 443–670 ) in COS-7 cells co-transfected with EB1-GFP (green). The bar graph showed the averaged values of MT tip-to-cytosol intensity ratio under dark and lit conditions. n = 16 cells from three independent experiments. g , h Representative confocal images in HeLa cells ( g ) and quantification of the cytosol-to-PM translocation ( h ) of the indicated CRY2-PB variants before and after blue light illumination. n = 16 cells from three independent experiments. i – k Schematic showing light inducible ER-MT interactions in COS-7 cells cotransfected ER-anchored mCh-CRY2-STIM1 443–670 (red) with ( j ) EB1-GFP (green) or ( k ) GFP-tubulin (green). j Representative confocal images showing ER morphology change following blue light illumination. The mCherry and GFP fluorescent intensities across the dashed line were plotted next to the images to indicate the degree of signal overlaps. k Confocal images showing the clustering of ER-resident mCh-CRY2-STIM1 443–670 along with GFP-tubulin (green) upon blue light illumination. The surface profiles of a selected area (oval) were presented to aid the visualization of subcellular distribution. l , m Light-inducible assembly of ER-plasma membrane contact sites (MCSs) mediated by ER-resident mCh-CRY2-PB (STIM1 671–685 ). l Schematic of the design. m Representative confocal images of the footprint of HeLa cells transfected with mCh-CRY2-PB variants before and after blue light illumination.

Article Snippet: To add photosensitive domains into the cytoplasmic domain of human STIM1 (hSTIM1 233–685 ), we first amplified the iLID (LOV2-ssrA) and sspB components from the templates pLL7.0-Venus-iLID-Mito (Addgene; #60413) and pQE-80L-MBP-sspB-Nano (Addgene; #60409), and then inserted them into mCh-STIM1 233–685 (pmCherry-C1) or YFP-STIM1 233–685 (pEYFP-C1) with a flexible linker (SGGGGGGG) 3 to obtain mCh/YFP-tagged iLID-STIM1 233–685 or sspB-STIM1 233–685 .

Techniques: Binding Assay, Translocation Assay, Transfection

Journal: PLoS ONE

Article Title: Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

doi: 10.1371/journal.pone.0055183

Figure Lengend Snippet: (A) Cells were treated with 50 µM of resveratrol for 12 h and stained with propidium iodide. DNA content was analysed by flow cytometry. The representative results of three independent experiments are shown. (B) Results are the mean of data, with error bars representing the SD of triplicate values. (C) Cells were treated with 50 µM of resveratrol for 24, 36 and 48 h. 50 µg of whole cell extract was prepared for each cell line, separated with SDS-PAGE and subjected to Western blotting with antibodies specific to cdc2, cdk2 and cdk3. The blots were stripped and reprobed with α-tubulin antibody to show equal protein loading. The figures shown are representative results of three independent experiments.* P <0.05.

Article Snippet: Anti-cdk3 antibody was obtained from Genetex (San Antonio, Texas).

Techniques: Staining, Flow Cytometry, SDS Page, Western Blot