Journal: Journal of Biological Chemistry

Article Title: The LIM-only Protein FHL2 Regulates Cyclin D1 Expression and Cell Proliferation

doi: 10.1074/jbc.m800708200

Figure Lengend Snippet: FIGURE 1. FHL2 is associated with the cyclin D1 promoter. A, analysis of cyclin D1 transcripts in three independent WT and FHL2/ immortalized clones by real time RT-PCR. Cyclin D1 expression was normalized to 18 S RNA. The ratio of the cyclin D1/18 S signal in WT clone B was arbitrarily set at 1. The average S.D. values and for three independent experiments are shown. Wild type clones are indicated by capital letters, and FHL2/clonesareshownbylowercaseletters.B,schematicrepresentationofAP1andTCF/CREelementsatthe cyclin D1 promoter. C, association of FHL2 with the cyclin D1 promoter at the TCF/CRE site. WT and FHL2/ MEFs were analyzed by ChIP. Antibodies against FHL2 and HA were used in parallel immunoprecipitations. Eluted DNA was amplified by PCR using cyclin D1 primers F1-R1 and F2-R2 indicated in B and control primers of the cad promoter (see “Experimental Procedures”). Data are representative of three independent experiments. D, graphic representation of the data of real time PCR for the TCF/CREB binding site in immortalized wild type and FHL2/ MEFs. Calculation of the amount of immunoprecipitated DNA relative to that present in total input chromatin (percentage of total) is as follows: % total 2CT (where CT represents cycle threshold). CT CT (input) CT (FHL2IP). The total percentage at the 0 h time point of serum stimulation in WT MEFs was arbitrarily set at 1. Two independent immortalized FHL2/ clones were analyzed in two independent ChIP experiments.

Article Snippet: Retroviral Vectors and Gene Transfer—pBabe-FHL2 was constructed by inserting full-length FHL2 cDNA in the pBabepuro vector. pBabe puro cyclin D1 HA constructed by Dr. William Hahn was obtained from Addgene (Addgene plasmid 9050).

Techniques: Clone Assay, Quantitative RT-PCR, Expressing, Amplification, Control, Real-time Polymerase Chain Reaction, Binding Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: The LIM-only Protein FHL2 Regulates Cyclin D1 Expression and Cell Proliferation

doi: 10.1074/jbc.m800708200

Figure Lengend Snippet: FIGURE 4. Restoration of cyclin D1 and cell proliferation in FHL2/ MEFs by re-expression of FHL2. A, up-regulation of cyclins D1, D2, D3, and E and Rb phosphorylation in FHL2-restored cells. Upper panel, mRNA levels of cyclin D1 and FHL2 in immortalized MEFs of different genotypes. Real time RT-PCR was carried out using either cyclin D1- or FHL2-specific primers on total RNA extracted from indicated cell lines. Their expres- sion was normalized to 18 S RNA. The average and S.D. values for three independent experiments are shown. Data are representative of those from three independent clones for WT and FHL2/ genotypes and three independent pools of FHL2-restored and pBabe-infected cells. The ratio of cyclin D1/18 S or FHL2/18 S in WT MEFs was arbitrarily set at 1. In the lower panel, cell lysates were analyzed by immunoblotting. The asterisk shows hyperphosphorylated Rb. B, growth curves of immortalized WT, FHL2/, pBabe-infected and pBabe- FHL2-infected FHL2/ MEFs. The data presented are the mean S.D. obtained from three independent experiments. Data are representative of growth curves from two independent FHL2-infected MEF pools.

Article Snippet: Retroviral Vectors and Gene Transfer—pBabe-FHL2 was constructed by inserting full-length FHL2 cDNA in the pBabepuro vector. pBabe puro cyclin D1 HA constructed by Dr. William Hahn was obtained from Addgene (Addgene plasmid 9050).

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Clone Assay, Infection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The LIM-only Protein FHL2 Regulates Cyclin D1 Expression and Cell Proliferation

doi: 10.1074/jbc.m800708200

Figure Lengend Snippet: FIGURE5.EctopicexpressionofcyclinD1inFHL2/MEFs.A,expressionof cell cycle regulators in FHL2/ MEFs infected with pBabecyclin D1-HA. Increased cyclin D1 expression in FHL2/ MEFs after introduction of the cyclin D1-HA transgene was confirmed by immunoblotting with both anti- cyclin D1 and anti-HA antibodies. B, growth curves of immortalized wild type, FHL2/, pBabe-infected and pBabecyclin D1-HA-infected FHL2/ MEFs. Equal numbers of cells were plated for triplicate at the beginning of the experiment.Cellnumbersweredeterminedeverydayforatotalof6days.The data presented are the mean S.D. of values obtained from three independ- ent experiments. The results shown are representative of those from two independent cyclin D1-HA-expressing MEF pools.

Article Snippet: Retroviral Vectors and Gene Transfer—pBabe-FHL2 was constructed by inserting full-length FHL2 cDNA in the pBabepuro vector. pBabe puro cyclin D1 HA constructed by Dr. William Hahn was obtained from Addgene (Addgene plasmid 9050).

Techniques: Infection, Expressing, Western Blot

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: Discovery of MS28, the first cyclin D1 degrader. (A) Schematic representation of bridged PROTAC. The bridged PROTAC, which binds a bridge protein but not its binding partner (POI), recruits the protein complex into proximity with an E3 ligase, resulting in preferential polyubiquitination and subsequent degradation of the POI by the proteasome over the bridge protein. (B) Chemical structure of MS28. (C) Chemical structures of MS28N1 and MS28N2. (D) Western blot (WB) results of MS28, MS28N1, BSJ-03–204 (BSJ), MS140, and PB on degrading cyclin D1 and CDK4/6. Calu-1 cells were treated with the indicated compound at the indicated concentrations for 8 h. Results shown are representative of at least two independent experiments. (E) Binding affinities of MS28, MS28N1, MS28N2, and PB to CDK4 and CDK6 in biochemical assays. Results shown are the mean ± SD from duplicate experiments.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Binding Assay, Western Blot

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 preferentially degrades cyclin D1 over CDK4/6 in Calu-1 cells. (A) Left, MS28 degrades cyclin D1 first and CDK4/6 subsequently. Calu-1 cells were treated with DMSO or 3 μM of MS28 for the indicated time. Results shown are representative of three independent experiments. Right, quantification of relative cyclin D1, CDK4, and CDK6 abundance at each time point, following 3 μM MS28 treatment (four technical repeats from three biological repeats). P-values were calculated for each protein relative to its abundance at 0.5 h. ****P < 0.0001, ***P < 0.001, and **P < 0.01. The line above the bars indicates that all of the encompassed bars share the same p-value above the line. (B) MS28 concentration-dependently degrades cyclin D1. Calu-1 cells were treated with MS28 at the indicated concentration for 8 h. Results shown are representative of two independent experiments. (C) DC50 and Dmax values of MS28 in Calu-1 cells (calculated from the WB data in panel B and biological repeat). (D) MS28 did not change the mRNA levels of CCND1, CDK4, and CDK6 in RT-qPCR studies. Calu-1 cells were treated with DMSO, 3 μM MS28, or 3 μM PB for 4 h. mRNA levels are relative to DMSO control. Results are representative of two biological repeats.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Concentration Assay, Quantitative RT-PCR

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 effectively degrades cyclin D1 in a VHL-, CDK6-, and UPS-dependent manner. (A) Left, VHL KO rescues the MS28-induced degradation of cyclin D1 and CDK4/6. Calu-1 cells were transduced with lentivirus containing VHL-targeting sgRNA or an empty vector. Antibiotic-selected cells were checked for VHL expression (Top: WB results confirm CRISPR-mediated VHL KO in Calu-1 cells), followed by treatment with MS28 for 8 h. Right, quantification of cyclin D1 and CDK4/6 abundance in the indicated experimental groups. P-values for each protein were calculated between the mock and VHL KO groups (indicated by bar) within each concentration. (B) Left, CDK6 KD via siRNA rescues the MS28-induced degradation of cyclin D1. Calu-1 cells were transfected with CDK6-targeting siRNA for 2 days, followed by treatment with MS28 for 8 h. Right, quantification of cyclin D1 abundance in the indicated experimental groups. P-values were calculated between the control and CDK6 KD group within each MS28 concentration. (C) VHL coelutes with cyclin D1-CDK6 in the presence of MS28. His-tagged CDK6 was immobilized on cobalt agarose resin and incubated overnight along with cyclin D1. The VCB complex was added the next day with either DMSO or MS28. Pretreatment with MG132 (D) or MLN4924 (E) rescues the MS28-induced degradation of cyclin D1 and CDK4/6. Quantification of cyclin D1 and CDK4/6 abundance are listed aside. For each protein, p-values were calculated between groups indicated by the lines above the bars. Calu-1 cells were pretreated with MG132 or MLN4924 for 1 h, followed by treatment with MS28 for 8 h. WB results shown in panels A–E are representative of at least two independent experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Transduction, Plasmid Preparation, Expressing, CRISPR, Concentration Assay, Transfection, Incubation

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 is selective for cyclin D1/D3 and CDK4/6. (A) Left, MS28, but not MS28N1, degrades cyclin D1/3 and CDK4/6, and reduces the cyclin A2 level, but not cyclin D2/B1 and CDK2 in Calu-1 cells (4 h-treatment at 3 μM). WB data shown are representative of two biological repeats. Right, quantification of the relative abundance of cyclins and CDKs analyzed in the WB upon DMSO, MS28, or MS28N1 treatment. P-values were calculated relative to the DMSO control for each protein. ****P < 0.0001, ***P < 0.001, and **P < 0.01. (B) MS28 is selective for CDK6 over a panel of 57 other kinases at 1 μM concentration. Data are the means ± SD from duplicate experiments.

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Concentration Assay

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: MS28 effectively suppresses the downstream Rb-E2F pathway, proliferation, and tumorigenesis in cancer cells. (A) MS28 and PB significantly reduced mRNA levels of E2F target genes (CCNA2, CCNE1, CDC6, PCNA, and PLK1) in RT-qPCR studies. Calu-1 cells were treated with DMSO, 3 μM of MS28, or PB for 8 h. P-values were calculated in comparison to DMSO from two biological repeats. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. (B) MS28 inhibits cell growth much more effectively than PB, BSJ, and MS28N1 in Calu-1 cells (treated with the indicated compound for 5 days). Data shown are the mean values ± SD from two biological repeats (each with three technical repeats). (C) MS28 suppresses clonogenicity of Calu-1 cells in a soft agar assay more effectively than PB, BSJ, and MS28N1. Images were taken at the end of the 20-day treatment. Each treatment group (at 0.3 μM) is representative of three independent experiments, each with at least five technical repeats. (D) MS28, not MS28N1, BSJ, or PB, effectively degrades cyclin D1 in NCI-H2110 cells (treated with the indicated compound at the indicated concentrations for 8 h). Results are representative of two biological repeats. (E) MS28, but not MS28N1, BSJ or PB, potently inhibits the growth of NCI-H2110 cells (5 days treatment, three biological repeats).

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: Quantitative RT-PCR, Soft Agar Assay

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: Human Primer Sequences Used in RT-qPCR Studies

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Bridged Proteolysis Targeting Chimera (PROTAC) Enables Degradation of Undruggable Targets

doi: 10.1021/jacs.2c09255

Figure Lengend Snippet: sgRNA Sequences Used in CRISPR/Cas9-Mediated KO Studies

Article Snippet: Recombinant His-tagged CDK6 and GST-tagged cyclin D1 complex were purchased from BPS Bioscience (cat. # 40097), and recombinant human VCB complex was purchased from R&D Systems (cat. # E3–600).

Techniques: CRISPR, Sequencing