Journal: bioRxiv

Article Title: Retroelement decay by the exonuclease XRN1 is a viral mimicry dependency in cancer

doi: 10.1101/2023.03.30.531699

Figure Lengend Snippet: dsRNA induced by Palbociclib or 5-AZA-CdR produces a synthetic dependency to XRN1 in XRN1-resistant POP92 cells. (A) Dot blot for dsRNA using total RNA from indicated cell lines. Normalized amounts of total RNA were dotted on Hybond N+ membranes, visualized by methylene blue staining, and immunoblotted with J2 antibody. (B) Representative confocal microscopy images of colorectal cell lines. Nuclei were stained with DAPI (blue) and dsRNA was stained using the J2 antibody (red). (C) Representative confocal microscopy images from control and knockout of XRN1 of POP92 cells treated with PBS or 5-AZA-CdR. Nuclei were stained with DAPI (blue) and dsRNA was stained using the J2 antibody (red). (D) Survival of wild-type XRN1 (black) and XRN1 knockout (red) patient-derived CRC cells (POP92) after treatment with 5-AZA-CdR. Luminescence signal was normalized, and dose-response curves and EC50 values were calculated using a nonlinear regression curve fit. (E) Survival of wild-type XRN1 (black) and XRN1-knockout (red) POP92 cells after treatment with palbociclib. Luminescent signal was normalized, and dose-response curves and EC50 values were calculated using a nonlinear regression curve fit.

Article Snippet: Palbociclib isethionate (HY-A0065) was purchased from Med Chem Express.

Techniques: Dot Blot, Staining, Confocal Microscopy, Control, Knock-Out, Derivative Assay

Journal: bioRxiv

Article Title: Retroelement decay by the exonuclease XRN1 is a viral mimicry dependency in cancer

doi: 10.1101/2023.03.30.531699

Figure Lengend Snippet: Proposed mechanism for XRN1 dependent viral mimicry adaptation. A) XRN1 resistant cell lines have low levels of immunogenic dsRNA and are therefore not relying on XRN1 to resist viral mimicry induced cell death. XRN1 sensitive cell lines require XRN1 to resist high levels of immunogenic dsRNA to induce viral mimicry induced cell death. Viral mimicry inducing drugs such as 5-AZA-CdR and palbociclib can generate a synthetic dependency to XRN1. B) XRN1 degrades dsRNA while ADAR1 edits A-to-I in dsRNA, these mechanisms have different effects on activation of MDA5 and PKR pathways. When ADAR1 and XRN1 are not present, both MDA5 and PKR can bind dsRNA and activate viral mimicry. If XRN1 is knocked out and only ADAR1 is present, the edited dsRNA will not activate the MDA5 pathway while the PKR pathway can still be activated. If both XRN1 and ADAR1 is present, dsRNA is both edited and degraded which hinders activation of MDA5 and PKR pathways.

Article Snippet: Palbociclib isethionate (HY-A0065) was purchased from Med Chem Express.

Techniques: Activation Assay

Journal: Oncology reports

Article Title: Targeting P16INK4A in uterine serous carcinoma through inhibition of histone demethylation.

doi: 10.3892/or.2019.7067

Figure Lengend Snippet: Figure 1. Differential expression of P16INK4A in tumor samples. (A) Representative images of P16INK4A positivity in USC, P16INK4A negativity in UEC and P16INK4A negativity in normal endometrium. The normal tissue was obtained from 8 patients with leiomyoma who receiving hysterectomy procedures. Scale bar, 50 µm. (B) Percentage of P16INK4A‑positive cases among 33 USC and 88 UEC samples. **P<0.01 (χ2 test). (C) Western blot analysis of 6 endometrial cell lines (AN3CA, Hec1A, Hec108, Nou‑1, EFE‑184 and ETN‑1). Vinculin was used as a loading control. (D) The reverse transcription‑quantitative polymerase chain reaction results of the 6 endometrial cell lines (AN3CA, Hec1A, Hec108, Nou‑1, EFE‑184 and ETN‑1) were analyzed and shown as relative P16INK4A mRNA level of the Hec1A values, which were then converted as fold change. *P<0.05 and **P<0.01 [analysis of variance and a post hoc test (Student‑Newman‑Keuls)]. (E) Response of different endometrial cell lines to the CDK4/6 inhibitor palbociclib. The viability of ETN‑1, EFE‑184, Hec‑1A, AN3CA, Hec108 and Nou‑1 cells was measured with a Cell Counting Kit‑8 assay following treatment with vehicle (dimethyl sulfoxide) and palbociclib (0.5, 1, 2.5, 5 and 10 µM) for 72 h. The IC50 are depicted. P16INK4A, cyclin-dependent kinase inhibitor 2A; CDK4, cyclin-dependent kinase 4; UEC, uterine endometroid carcinoma; USC, uterine serous carcinoma; H3K27, histone 3 lysine 27; KDM6B, histone lysine demethylase 6B; IC50, half‑maximal inhibitory concentration; RB, retinoblastoma.

Article Snippet: Palbociclib was purchased from Selleck Chemicals (Houston, TX, USA; cat. no. S1579), and GSK-J4 was obtained from MedChemExpress (Shanghai, China; cat. no. HY-15648B).

Techniques: Quantitative Proteomics, Western Blot, Control, Polymerase Chain Reaction, CCK-8 Assay, Concentration Assay

Journal: Molecular cell

Article Title: Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein’s C-Terminal Helix

doi: 10.1016/j.molcel.2019.03.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Palbociclib , Santa Cruz Biotechnology , Cat#sc-478943.

Techniques: Purification, Produced, Virus, Recombinant, Selection, Membrane, Flow Cytometry, Expressing, Plasmid Preparation, Generated

Journal: Cell reports

Article Title: SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer

doi: 10.1016/j.celrep.2024.113924

Figure Lengend Snippet: (A and B) Cell (A) proliferation and (B) scratch-wound healing process of HuH-7 cell lines stably expressing WT HuR and the different SUMOylation mutants analyzed in the IncuCyte system. (C) Representative pictures of 3D spheroids from HuH-7 cell lines stably expressing WT HuR and the SUMOylation mutant species embedded on a collagen type I matrix for 48 h, and quantification of the relative invasive area. (D) Percentage of apoptosis detected in the WT HuR and the indicated SUMOylation mutant HuH-7 cell variants by flow-cytometry analysis after FITC-annexin V and viability stainings, relative to STS-treated HuH-7 cells. (E) Quantification of cell proliferation in the HuH-7 cell lines stably expressing WT HuR and the different SUMOylation mutants after an acute 3-day treatment with a range of palbociclib concentrations analyzed by crystal violet staining. (F) Quantification of relative senescence in the HuH-7 cell lines stably expressing the WT and the K120/182R, K120R, K182R HuR mutant species after chronic 2-week treatment with a range of palbociclib concentrations analyzed by β-galactosidase staining. (G) Crystal violet staining of colonies from HuH-7 cell lines stably expressing WT HuR and the SUMOylation mutants treated with the indicated doses of palbociclib for 10 days, and generation of dose-response curves for calculation of IC 50 values. In (A–F), data are presented as the mean ± SD of at least three biological replicates within one representative experiment. *p < 0.05, **p < 0.01, and ***p < 0.001, two-tailed t test vs. HuH-7 WT HuR . If not indicated otherwise, the differences were not significant. In (C) and (G), images are representative of at least three biological replicates within one representative experiment. See also – .

Article Snippet: Palbociclib, isethionate salt, >99% , LC Laboratories , Cat#P-7766.

Techniques: Stable Transfection, Expressing, Mutagenesis, Flow Cytometry, Staining, Two Tailed Test

Journal: Cell reports

Article Title: SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer

doi: 10.1016/j.celrep.2024.113924

Figure Lengend Snippet: (A) Enrichment of SUMOylated HuR in the HuH-7 WT HuR and HuH-7 HuR [K120/182R] cells treated with 5 nM palbociclib for 2 weeks by means of protein pull-down with GST control and SUBEs in combination with western blotting analysis. (B and C) (B) SUMO1 and (C) SUMO2/3 protein expression levels and quantification in the HuH-7cell lines stably expressing WT HuR and the K120/182R, K120R, K182R HuR SUMOylation mutant species treated with 5 nM palbociclib for 2 weeks. In (A–C), western blots are representative of at least three biological replicates within one representative experiment. In (B) and (C), data are presented as the mean ± SD of at least three biological replicates within one representative experiment. *p < 0.05 and **p < 0.01, two-tailed t test vs. 0 nM palbociclib or HuH-7 WT HuR . If not indicated otherwise, the differences were not significant.

Article Snippet: Palbociclib, isethionate salt, >99% , LC Laboratories , Cat#P-7766.

Techniques: Control, Western Blot, Expressing, Stable Transfection, Mutagenesis, Two Tailed Test

Journal: Cell reports

Article Title: SUMOylation controls Hu antigen R posttranscriptional activity in liver cancer

doi: 10.1016/j.celrep.2024.113924

Figure Lengend Snippet:

Article Snippet: Palbociclib, isethionate salt, >99% , LC Laboratories , Cat#P-7766.

Techniques: Recombinant, Protease Inhibitor, Mutagenesis, Transfection, Purification, Staining, Luminescence Assay, Colorimetric Assay, Sequencing, Plasmid Preparation, Software, Microscopy

Journal: Molecular & Cellular Proteomics : MCP

Article Title: The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint

doi: 10.1074/mcp.RA120.002069

Figure Lengend Snippet: High resolution chemical proteomics reveals proteins whose abundances are APC/C regulated. A , Workflow for the chemical proteomics experiment described in this study. Asynchronous RPE1 cells were arrested in 1 μ m palbociclib (a Cdk4/6 inhibitor) for 20 h, at which point they were acutely treated with either DMSO or a combination of 6 μ m proTAME + 50 μ m apcin (referred to as “APC/C inhibitors” or “APC/Ci”), in the ongoing presence of palbociclib. Cells were then collected at time 0 (the time of APC/C inhibitor addition) or 8 h after drug addition and were harvested for TMT-based proteomic identification and quantification. Samples were analyzed in biological triplicate within a 10-plex TMT label set, with the 10th channel used as a bridge. B , Asynchronous RPE1 cells were treated with either DMSO or 1 μ m palbociclib for 20 h. Cells were harvested, and lysates were analyzed by immunoblot for the indicated proteins. C , Previously reported APC/C substrates that were identified in this study are plotted with their observed fold change in the APC/C inhibitor treated sample (APC/Ci) relative to the DMSO treated sample. Error bars represent the standard deviation (S.D.) between the three biological replicates measured by MS. Asterisks indicate an abundance increase over control that is statistically significant (*: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001) D , Previously reported APC/C substrates that were identified as increasing by MS in G 1 RPE1 cells treated with APC/C inhibitors were validated by immunoblot for selected proteins. E , Volcano plot highlighting all published APC/C substrates identified in this study ( blue ) as well as proteins that (1) contain a high probability D- and/or KEN-box ( d -box = green , KEN-box = pink , D- and KEN-boxes = purple ), (2) increase ≥1.15-fold under APC inhibition, (3) were identified by >1 peptide, and (4) have a p -value < 0.05.

Article Snippet: The following chemicals were used: palbociclib (LC Laboratories, P-7722), proTAME (Boston Biochem, I-440), MG132 (474790, Calbiochem), S-trityl l -cysteine (STLC, Alfa Aesar, L14384), thymidine (Sigma Aldrich, T9250), nocodazole (Sigma Aldrich, 31430-18-9), RO3306 (AdipoGen Life Sciences, AGCR13515M), doxycycline hyclate (Sigma Aldrich, D9891).

Techniques: Western Blot, Standard Deviation, Control, Inhibition

Journal: Molecular & Cellular Proteomics : MCP

Article Title: The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint

doi: 10.1074/mcp.RA120.002069

Figure Lengend Snippet: IRS2 levels are controlled by Cdh1 in a proteasome-dependent manner. A , Cells were treated identically to what is described in A , and IRS2 abundance was measured by immunoblot. B , C 2 C 12 myoblasts were induced to differentiate through serum withdrawal and supplementation with insulin, transferrin, and selenium (ITS). After 3 days of differentiation, myotubes were acutely treated with either DMSO or APC/C inhibitors. After eight hours of drug treatment, myotubes were collected and IRS2 levels from all samples were analyzed by immunoblotting. C – D , Asynchronous HeLa ( C ) and RPE1 ( D ) cells were transfected with either a control or Cdh1-directed siRNA for 24 h. Cells were allowed to grow for an additional 24 h before collection and analysis of the indicated protein levels in lysate by immunoblot. E , HeLa cells were mock transfected or transfected with increasing amounts of a plasmid encoding myc-tagged human Cdh1 for 24 h. Cells could grow for an additional 24 h before collection and analysis of the indicated protein levels by immunoblot. F , RPE1 cells were arrested in G 1 with 1 μ m palbociclib for 20 h. Following G 1 arrest, cells were treated with DMSO, APC/C inhibitors, MG132, or a combination of APC/C inhibitors and MG132 for an additional 8 h. Cells were harvested, and lysates were analyzed by immunoblot for IRS2 abundance. G , 6xHis-tagged ubiquitin conjugates were isolated from HeLa cell lysates using Ni-NTA agarose resin. Lysates were derived from cells expressing 6xHis-ubiquitin and HA-tagged IRS2 that were treated with MG132 alone or in combination with APC/C inhibitors. Resin eluate and inputs were probed by immunoblot using an HA antibody, and Ponceau staining was used as a loading control.

Article Snippet: The following chemicals were used: palbociclib (LC Laboratories, P-7722), proTAME (Boston Biochem, I-440), MG132 (474790, Calbiochem), S-trityl l -cysteine (STLC, Alfa Aesar, L14384), thymidine (Sigma Aldrich, T9250), nocodazole (Sigma Aldrich, 31430-18-9), RO3306 (AdipoGen Life Sciences, AGCR13515M), doxycycline hyclate (Sigma Aldrich, D9891).

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Ubiquitin Proteomics, Isolation, Derivative Assay, Expressing, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint

doi: 10.1074/mcp.RA120.002069

Figure Lengend Snippet: Cdh1's ability to control IRS2 levels depends on a C-terminal d -box motif. A , ( top ) Schematic depicting IRS2's protein domain structure. PH = pleckstrin homology domain, PTB = phosphotyrosine binding domain, KRLB = kinase regulatory-loop binding region. IRS2's C-terminal full d -box motif is highlighted in red. ( bottom ) Comparison of IRS2's D-box conservation among amniotes. B , RPE1 cells stably expressing lentivirus-derived, doxycycline-inducible, C-terminally HA-tagged IRS2 constructs were arrested in G 1 with palbociclib for 20 h. Following arrest, samples were either collected or DMSO or APC/C inhibitors were added for an additional 8 h. Quantification of immunoblots shown at right: HA levels were normalized to a loading control and are plotted relative to DMSO levels. Error bars = mean ± S.E. *: p = 0.0187; ns: p = 0.816. C , C 2 C 12 myoblasts stably expressing lentivirus-derived, doxycycline-inducible, C-terminally HA-tagged IRS2 constructs were grown to confluence and switched to low serum media supplemented with ITS (differentiation media) and doxycycline. Cells were allowed to differentiate into myotubes for 3 days (with media refreshment every 24 h), at which point (0 h) either DMSO or APC/C inhibitors for an additional 8 h in the presence of doxycycline. Quantification of immunoblots shown at right: HA levels were normalized to a loading control and are plotted relative to DMSO levels. Error bars = mean ± S.E. *: p = 0.0118; ns: p = 0.910. D , Asynchronous RPE1 cells stably expressing lentivirus-derived, doxycycline-inducible C-terminally HA-tagged IRS2 constructs were transfected with a nontargeting (control) siRNA or an siRNA directed against Cdh1 for 24 h in the absence of doxycycline. Quantification of immunoblots shown at right: HA levels were normalized to a loading control and are plotted relative to DMSO levels. Error bars = mean ± S.E. *: p = 0.0132; ns: p = 0.963. E , Asynchronous HeLa cells stably expressing lentivirus-derived, N-terminally FLAG-HA tagged IRS2 constructs were transfected with a nontargeting (control) siRNA or an siRNA directed against Cdh1 for 24 h. Quantification of immunoblots shown at right: HA levels were normalized to a loading control and are plotted relative to DMSO levels. Error bars = mean ± S.E. *: p = 0.0131; ns: p = 0.803. F , Comparison of the Hs IRS2 D-box sequence with the corresponding region from Hs IRS1. G , MS-quantified IRS1 and IRS2 abundance in G 1 APC inhibitor proteomics . IRS1 abundance was quantified based on 5 peptides (4 unique) in 3 biological replicates; IRS2 was quantified based on 3 peptides (all unique) in 3 biological replicates. H , RPE1 cells were subject to the same conditions described in A , and cell lysates were analyzed by immunoblotting for IRS1 abundance I , RPE1 cells were treated as in C . Cell lysates were analyzed by immunoblotting for IRS1 abundance.

Article Snippet: The following chemicals were used: palbociclib (LC Laboratories, P-7722), proTAME (Boston Biochem, I-440), MG132 (474790, Calbiochem), S-trityl l -cysteine (STLC, Alfa Aesar, L14384), thymidine (Sigma Aldrich, T9250), nocodazole (Sigma Aldrich, 31430-18-9), RO3306 (AdipoGen Life Sciences, AGCR13515M), doxycycline hyclate (Sigma Aldrich, D9891).

Techniques: Control, Binding Assay, Comparison, Stable Transfection, Expressing, Derivative Assay, Construct, Western Blot, Transfection, Sequencing