Journal: The Journal of Clinical Investigation

Article Title: RSK1-driven TRIM28/E2F1 feedback loop promotes castration-resistant prostate cancer progression

doi: 10.1172/JCI185119

Figure Lengend Snippet: ( A and B ) C4-2B and DU145 cells treated with RSK inhibitors were analyzed by immunoblot. ( C and D ) C4-2B with Rb knockdown and DU145 cells treated by vehicle (Veh), 500 nM palbociclib (Pal), 1 μM BI-D1870 (BI), and 20 μM LJH685 (LJH) were subjected to the colony formation assay. Quantification was conducted by image J and presented as mean ± SEM, n = 3. Two-tailed unpaired Student’s t test, with the Holm-Bonferroni method applied to correct for multiple comparisons. ** P < 0.01, *** P < 0.001. ( E and F ) PCa organoids were generated from prostate tumors in Pb-Cre: Pten –/– mice. ( E ) Representative images were shown. ( F ) Quantification was presented as mean ± SEM, n = 3. Two-tailed unpaired Student’s t test, with the Holm-Bonferroni method applied to correct for multiple comparisons. *** P < 0.001. ( G ) The experimental design of LuCaP145.1 PDX assay and treatment strategy. ( H – J ) NGS mice were implanted subcutaneously with LuCaP 145.1 tumors. Mice were randomized and treated with vehicle, palbociclib (75 mg/kg) and BI-D1870 (50 mg/kg) for 3 weeks. Tumor sizes were plotted against days of treatment ( H ). The tumor volume data were presented as mean ± SEM, n = 5. The statistical analysis was performed using a 1-way ANOVA with Tukey’s HSD test for multiple comparisons P value adjustment. *** P < 0.001. Tumor weight was presented as boxplot ( I ), and the toxicity was evaluated by mouse body weight ( J ). The data was presented as mean ± SEM, n = 5. Two-tailed unpaired Student’s t test, with the Holm-Bonferroni method applied to correct for multiple comparisons. * P < 0.05. ( K – N ) Tumor tissues were subjected to IHC assay. Magnification ×20. ( K ), followed by quantification ( L – N ). The data was presented as mean ± SEM, n = 5. Two-tailed unpaired Student’s t test, with the Holm-Bonferroni method applied to correct for multiple comparisons. *** P < 0.001.

Article Snippet: Puromycin (P8833) and doxycycline (D9891) were from Sigma-Aldrich, and G418 (T6512), ulixertinib (T7005), BI-D1870 (T6171), palbociclib (T6239), and LJH685 (T6877) were from Targetmol.

Techniques: Western Blot, Knockdown, Colony Assay, Two Tailed Test, Generated

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: 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: Molecular cell

Article Title: Synthetic lethal and resistance interactions with BET bromodomain inhibitors in triple-negative breast cancer

doi: 10.1016/j.molcel.2020.04.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Palbociclib , MedChem Express , HY-50767A.

Techniques: Recombinant, CRISPR, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The CDK4/6 inhibitors (Palbociclib, HY-50767S, MedChemExpress, China, 200 mg per kg bodyweight, oral administration) and RBPJ Inhibitor-1 (RIN1, HY-137471, MedChemExpress, China, 40 mg per kg bodyweight, intraperitoneal injection) were used for mice study.

Techniques: Expressing

Journal: Analytical Chemistry

Article Title: β-Galactosidase-Activatable Nile Blue-Based NIR Senoprobe for the Real-Time Detection of Cellular Senescence

doi: 10.1021/acs.analchem.2c04766

Figure Lengend Snippet: (a) Images of control 4T1 (up) and senescent 4T1 cells (4T1 cells treated with palbociclib) (down). (b) Images of control SK-Mel-103 (up) and senescent SK-Mel-103 cells (SK-Mel-103 cells treated with palbociclib) (down). From left to right: X-Gal staining images and confocal images from nontreated cells, incubated with NBGal (1.25 μM) and NB (1.25 μM), respectively. (c) Quantification of the fluorescence emission intensity relative to the cell surface of control and senescent palbociclib-treated 4T1 cells and (d) of control and senescent palbociclib-treated SK-Mel-103 cells, respectively. The results exhibited representative data from three independent studies ( n = 3), and values are expressed as mean ± SD. Statistical analysis was assessed by applying two-way analysis of variance (ANOVA) with multiple comparisons (**** p < 0.001).

Article Snippet: The induction of senescence after palbociclib treatment in SK-Mel-103 and 4T1 cells was assessed using a Senescence β-Galactosidase Staining Kit (#9860, Cell Signaling) following the manufacturer′s instructions.

Techniques: Control, Staining, Incubation, Fluorescence

Journal: Analytical Chemistry

Article Title: β-Galactosidase-Activatable Nile Blue-Based NIR Senoprobe for the Real-Time Detection of Cellular Senescence

doi: 10.1021/acs.analchem.2c04766

Figure Lengend Snippet: (a) Palbociclib-induced senescent cancer model. For tumor generation, 4T1 cells were injected subcutaneously into the left mammary fat pad of female BALB/cByJ mice. Mice were subsequently treated with palbociclib (100 mg/kg) or vehicle by daily oral gavage (og) for 7 days. Mice were then divided into four groups of 5 animals: (A) Vehicle; (B) Vehicle + NBGal ; (C) palbociclib + NBGal ; and (D) palbociclib. NBGal was intravenously injected into the tail vein, and mice were monitored by IVIS imaging. Then, mice were sacrificed, and organs and tumors were collected. (b) Representative IVIS images of 4T1 tumor-bearing mice at 0.5 and 3 h post-injection. From left to right: (B) Vehicle + NBGal ; (C) palbociclib + NBGal ; and (D) palbociclib. (c) Quantification of average radiance intensity from IVIS images in the tumor zone shown in (b). The results are expressed as mean ± SD, and statistical analysis was performed by applying Two-way ANOVA with multiple comparisons (** p < 0.01 and **** p < 0.001).

Article Snippet: The induction of senescence after palbociclib treatment in SK-Mel-103 and 4T1 cells was assessed using a Senescence β-Galactosidase Staining Kit (#9860, Cell Signaling) following the manufacturer′s instructions.

Techniques: Injection, Imaging

Journal: Analytical Chemistry

Article Title: β-Galactosidase-Activatable Nile Blue-Based NIR Senoprobe for the Real-Time Detection of Cellular Senescence

doi: 10.1021/acs.analchem.2c04766

Figure Lengend Snippet: (a) Representative images of tumors from BALB/cByJ stained for SA-β-Gal activity and the proliferative markers Ki67 in mice treated with vehicle (left) or palbociclib (right). (b) Representative IVIS images of organs and tumors from mice treated with vehicle + NBGal (group B), palbociclib + NBGal (group C), and palbociclib (group D). From left to right and up to bottom: lungs, liver, tumor, kidneys, spleen, and bladder. (c) Quantification of IVIS images shown in (b). The results are expressed as mean ± SD ( n = 5), and statistical analysis was performed by applying Two-way ANOVA with multiple comparisons (**** p < 0.001).

Article Snippet: The induction of senescence after palbociclib treatment in SK-Mel-103 and 4T1 cells was assessed using a Senescence β-Galactosidase Staining Kit (#9860, Cell Signaling) following the manufacturer′s instructions.

Techniques: Staining, Activity Assay

Journal: Oncogenesis

Article Title: CDK4/6 inhibitors and the pRB-E2F1 axis suppress PVR and PD-L1 expression in triple-negative breast cancer

doi: 10.1038/s41389-023-00475-1

Figure Lengend Snippet: A Immunoblot analysis showing the effect of 2-day palbociclib treatment on ngPVR and gPVR expression. B , C Immunoblot analysis of PD-L1 in TNBC lines treated with increasing ( B ) or high ( C ) concentrations of palbociclib for 2 days. D Immunoblot analysis of PD-L1 in TNBC lines with stable knockdown of RB1 , treated with/without palbociclib for 2 days. E Immunoblot analysis of PD-L1 in livers of mice on mixed background treated with palbociclib (140 mg/kg; gavage; 5 consecutive days/week) for 28 days and probed with AF1019 antibody (left) or in livers of C57BL/6 mice treated with palbociclib (140 mg/kg; gavage) for 7 consecutive days, probed with MAB90781 antibody (right). Bottom, PD-L1 quantification. Pure palbociclib (FA65120) was suspended in sodium lactate (50 nM, PH 4.0), which was used in the vehicle control mice. F Heatmap analysis of RNA-seq data (GSE177054) of MDA231 treated with ribociclib or ribociclib/D4476 for 2 days. White boxes denote out-of-range intensity. G Immunoblot analysis of PD-L1 in indicated tumor cells following knockdown of SPOP by RNAi over 2 (D2) or 3 (D3) days. H Immunoblot analysis of PD-L1 in indicated TNBC lines with/without SPOP knockdown after 2 days (D2) and treated with/without palbociclib for 24 h. I Immunoblot analysis of PD-L1 and SPOP in indicated TNBC lines after palbociclib treatment for 48 h. Numbers above immunoblots denote band intensity normalized to loading control.

Article Snippet: In vitro: pure palbociclib (Carbosynth, cat. FA65120) was solubilized in HCl in equal molar ratio.

Techniques: Western Blot, Expressing, Knockdown, Control, RNA Sequencing

Journal: Oncogenesis

Article Title: CDK4/6 inhibitors and the pRB-E2F1 axis suppress PVR and PD-L1 expression in triple-negative breast cancer

doi: 10.1038/s41389-023-00475-1

Figure Lengend Snippet: A Immunoblot analysis of PD-L1 in TNBC lines seeded at various densities in 6-cm plates and treated with 1 µM palbociclib for ~2 days. Bottom, PD-L1 quantification of ( A ) and Supplementary Fig. . Statistics calculated by pairwise analysis of seeding densities and treatment durations. B Immunoblot analysis of TNBC lines treated with various concentrations of hydrochloric acid (HCl) for 2 days. Right, PD-L1 quantification and analysis by Welch’s t -test. C Immunoblot analysis of MDA231 treated with/without palbociclib and/or with/without indicated solvents. D Immunoblot analysis of PD-L1 and PVR in different TNBC lines treated with physiological concentrations of lactic acid for 2 days. Numbers above immunoblots denote band intensity normalized to loading control. E A model suggesting that in response to mitogenic signals, CDK4/6 controls PD-L1 turnover by inducing its degradation via Culin3 SPOP and its transcription via pRB-E2F1. PD-L1 in red and green depicts old or newly synthesized glycosylated protein, respectively. F A model suggesting that the CDK4/6-RB-E2F axis modulates immune surveillance by transcriptionally regulating PD-L1, PVR and potentially other IC modulator genes. Solid and segmented arrows indicate validated (herein) and unvalidated targets, respectively; single arrows denote pRB and E2F1 binding sites as well as strong E2F1 recruitment by ChIP-seq analysis, whereas double arrows denote no or weak E2F1 recruitment.

Article Snippet: In vitro: pure palbociclib (Carbosynth, cat. FA65120) was solubilized in HCl in equal molar ratio.

Techniques: Western Blot, Control, Synthesized, Binding Assay, ChIP-sequencing