Journal: Current Oncology

Article Title: Prostate Cancer Biomarkers with a Focus on Galectin-3: Emerging Clinical and Therapeutic Implications

doi: 10.3390/curroncol33050280

Figure Lengend Snippet: Compartment-specific functions of Galectin-3 and therapeutic targeting strategies in prostate cancer.

Article Snippet: Melanoma & HNSCC , Immune Escape: Gal-3 acts as a negative immune checkpoint, blocking anti-PD-1 antibody binding to T-cells. , Belapectin + Pembrolizumab , Small molecule Galectin-3 inhibitor , Phase I study showed objective response rates of 50% (melanoma) and 33% (HNSCC). Demonstrates that Gal-3 inhibitors may enhance immunotherapy by reversing immune suppression [ , ]. .

Techniques:

Journal: Science signaling

Article Title: Aberrant Rac1-cofilin signaling mediates defects in dendritic spines, synaptic function, and sensory perception in fragile X syndrome

doi: 10.1126/scisignal.aan0852

Figure Lengend Snippet: (A to D) Representative Western blots and summary data for p-Thr508-LIMK (A), p-Ser9780-Slinghot1 (B), p-Ser3-cofilin (C), and the F-actin/G-actin ratio (D) in somatosensory synaptosomes from 1-week-old male WT and Fmr1 KO mice given a single subcutaneous injection of FRAX486 or vehicle [20% (w/v) hydroxypropyl-β-cyclodextrin] 8 hours before sacrifice (WT vehicle, n ≥ 12; KO vehicle, n ≥ 10;WT FRAX486, n ≥ 8;KO FRAX486, n ≥ 9). We note that overall there was no significant difference in total actin abundance in synaptosomes from FRAX486- versus vehicle-treated mice. Data are means ± SEM. *P < 0.05, **P < 0.01. (E) Schematic of Rac-cofilin signaling depicting proposed mechanism by which inhibition of group 1 PAKs (PAK1, PAK2, and PAK3) restores the abundance of p-LIMK1, p-Slingshot1, p-cofilin, and actin polymerization in Fmr1 KO animals.

Article Snippet: PAK inhibition The small-molecule inhibitor FRAX486 (Tocris Bioscience), which provides good potency and selectivity of group I PAKs (PAK1/2/3) over group II PAKs (PAK4/5/6) ( 43 ), was dissolved in 20% (w/v) hydroxypropyl-β-cyclodextrin vehicle (Sigma-Aldrich).

Techniques: Western Blot, Injection, Inhibition

Journal: Science signaling

Article Title: Aberrant Rac1-cofilin signaling mediates defects in dendritic spines, synaptic function, and sensory perception in fragile X syndrome

doi: 10.1126/scisignal.aan0852

Figure Lengend Snippet: (A) Representative evoked EPSC recordings from male WT (left) and Fmr1 KO (right) mice, either vehicle-treated (top) or FRAX486-treated (bottom) at P7. AMPAR-mediated EPSCs were measured as the peak current at −70 mV, and the NMDA component was measured by depolarizing the cell to +40 mV and measuring the current 60 ms after the onset of the outward current in the presence of 50 μM picrotoxin. Calibration: 100 ms, 50 pA. (B) Summary data of the NMDA/AMPA ratio in all recordings (WT vehicle, n = 13; Fmr1 KO vehicle, n = 11; WT FRAX486, n = 11; Fmr1 KO FRAX486, n = 6). (C) Representative traces of mEPSC recordings from male WT and Fmr1 KO animals, vehicle- or FRAX486-treated at P7. Calibration: 500 ms, 50 pA. (D) Summary data of mEPSC amplitude and frequency in all recordings (WT vehicle, n = 10; Fmr1 KO vehicle, n = 10; WT FRAX486, n = 11; Fmr1 KO FRAX486, n = 9). (E) Summary data for all recordings at P15 (NMDA/AMPA ratio: WT vehicle, n = 15; Fmr1 KO vehicle, n = 7; WT FRAX486, n =11; Fmr1 KO FRAX486, n = 10; mEPSC amplitude and frequency: WT vehicle, n = 18; Fmr1 KO, n = 7; WT FRAX486, n = 16; Fmr1 KO FRAX486, n = 12). Data are means ± SEM. *P < 0.05, **P < 0.01.

Article Snippet: PAK inhibition The small-molecule inhibitor FRAX486 (Tocris Bioscience), which provides good potency and selectivity of group I PAKs (PAK1/2/3) over group II PAKs (PAK4/5/6) ( 43 ), was dissolved in 20% (w/v) hydroxypropyl-β-cyclodextrin vehicle (Sigma-Aldrich).

Techniques:

Journal: Science signaling

Article Title: Aberrant Rac1-cofilin signaling mediates defects in dendritic spines, synaptic function, and sensory perception in fragile X syndrome

doi: 10.1126/scisignal.aan0852

Figure Lengend Snippet: (A) Timeline of the whisker-dependent texture discrimination task. (B) Discrimination of a novel versus familiar texture in WT and Fmr1 KO mice, as assessed by a preference index at chance level indicating impaired sensory processing (WT, n = 13; KO, n = 7). (C and D) Discrimination of a novel texture versus a familiar texture WT mice subjected to (C) whisker trimming (n = 10 animals) or (D) a texture-less object (n = 7 animals), indicating the necessity for an intact mystacial vibrissae to discriminate textures. (E) Novel object recognition task in WT and Fmr1 KO mice (WT, n = 7; KO, n = 8 animals). (F) Sensory processing, as assessed by the ability to discriminate a novel texture versus a familiar texture, in Fmr1 KO animals subjected to chronic administration of FRAX486 (once at P7, once at P14, and once at 3 to 4 weeks of age 24 hours before testing) (WT vehicle, n = 15; KO vehicle, n = 9; WT FRAX486, n = 10;KO FRAX486, n =10). Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: PAK inhibition The small-molecule inhibitor FRAX486 (Tocris Bioscience), which provides good potency and selectivity of group I PAKs (PAK1/2/3) over group II PAKs (PAK4/5/6) ( 43 ), was dissolved in 20% (w/v) hydroxypropyl-β-cyclodextrin vehicle (Sigma-Aldrich).

Techniques: Whisker Assay

Journal: Scientific Reports

Article Title: Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo

doi: 10.1038/s41598-019-50140-0

Figure Lengend Snippet: Enhanced efficacy of PTC-209 in combination with palbociclib against MDA-MB-231 cells. MDA-MB-231 cells were treated with the indicated dose of PTC-209 (a) and palbociclib (b) and cell viability was measured using alamarblue assay on day 4. Suppression of BMI1, CDK4, Rb, and pRb Ser795 is shown in upper panels. (c) Viability of MDA-MB-231 cells when treated with PTC-209 and palbociclib as single agent or in combination. Data are presented as mean ± S.E.M., n = 6. (d) Clonogenic assay showing colony forming capability of MDA-MB-231 treated with PTC-209, palbociclib, or combination of both at 5.0 µM. Plates were stained with Diff-Quik stain set on day 10. Data are representative of two independent experiments for each condition. Effect of PTC-209 and palbociclib, as single agents or in combination on MDA-MB-231 sphere formation (e) and cell migration using transwell migration (f) and scratch (g) assay. The number of migrating cells is indicated. Cell cycle analysis demonstrating the effects of PTC-209 and palbociclib as single agents or in combination on cell cycle distribution in MDA-MB-231 cells ( h , upper panel). Quantification of cell cycle distribution is shown in the lower panel (n = 3). (i) Representative fluorescence images of MDA-MB-231 cells treated with PTC-209 and palbociclib as single agents or in combination (5.0 µM). Cells were stained with acridine orange/ethidium bromide to detect apoptotic (cells with green condensed chromatin) and necrotic (red) cells.

Article Snippet: PTC-209 and palbociclib HCL small molecule inhibitors were purchased from Selleckchem (Houston, TX, USA).

Techniques: Alamar Blue Assay, Clonogenic Assay, Staining, Diff-Quik, Migration, Cell Cycle Assay, Fluorescence

Journal: Scientific Reports

Article Title: Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo

doi: 10.1038/s41598-019-50140-0

Figure Lengend Snippet: Effects of PTC-209 in combination with palbociclib against HCT116 and PC-3 cancer models. ( a) Cell viability of HCT116 (left) and PC-3 (right) treated with PTC-209 and palbociclib as single agent or in combination. Data are presented as mean +/− S.E.M., n = 6. (b) Representative microscopic images of HCT116 and PC-3 cells under different experimental conditions. (c) Clonogenic assay showing colony forming capability of HCT116 (left) cells treated with PTC-209 (0.6 µM), palbociclib (1.25 µM), or combination of both at (0.6 µM + 1.25 µM). Similarly, PC-3 cells (right) were treated PTC-209 (0.6 µM), palbociclib (0.6 µM), or combination of both at (0.6 µM + 0.6 µM). Plates were stained with Diff-Quik stain set on day 10. Wells are representative of two independent experiments for each treatment condition. (d) Effect of PTC-209 and palbociclib, as single agents or in combination on HCT116 and PC-3 cell migration using scratch assay at the above mentioned dose. (e) Quantification of cell cycle distribution in HCT116 (left) and PC-3 (right) cells treated with PTC-209, palbociclib, or combination of both at the above mentioned dose on day3.

Article Snippet: PTC-209 and palbociclib HCL small molecule inhibitors were purchased from Selleckchem (Houston, TX, USA).

Techniques: Clonogenic Assay, Staining, Diff-Quik, Migration, Wound Healing Assay

Journal: Scientific Reports

Article Title: Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo

doi: 10.1038/s41598-019-50140-0

Figure Lengend Snippet: Palbociclib predominantly target pathways regulating cell cycle and cell proliferation. ( a) Hierarchical clustering of MDA-MB-231 cells treated with palbociclib compared to vehicle-control treated cells based on differentially expressed mRNA levels using microarray analysis. Each column represents one replica and each row represents a transcript. Expression level of each gene in a single sample is depicted according to the colour scale. (b) Pie chart illustrating the distribution of the top 10 pathway designations for the differentially expressed genes in palbociclib compared to vehicle-control treated MDA-MB-231 cells. The pie size corresponds to the number of matched entities. (c) Expression levels of selected genes from the microarray data were validated using qRT-PCR. Data are presented as the means ± S.E.M., n = 6. **p < 0.01; ***p < 0.001. (d) Disease and function heat map depicting enrichment in the indicated functional and disease categories in the differentially expressed transcripts in palbociclib treated MDA-MB-231 cells based on IPA analysis. Colour scale indicates the activation score. (e) Bar chart illustrating the significantly enriched functional and disease categories (activation Z score ≤ or ≥2.0). Heat map-illustrating enrichment in the cell cycle (f) or cell death and survival (g) functional categories.

Article Snippet: PTC-209 and palbociclib HCL small molecule inhibitors were purchased from Selleckchem (Houston, TX, USA).

Techniques: Control, Microarray, Expressing, Quantitative RT-PCR, Functional Assay, Activation Assay

Journal: Scientific Reports

Article Title: Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo

doi: 10.1038/s41598-019-50140-0

Figure Lengend Snippet: Multiple upstream-regulated networks affected by palbociclib and PTC-209. ( a) Bar graph plot depicting the top 10 activated and top 10 inhibited upstream networks based on differentially expressed genes in MDA-MB-231 cells treated with palbociclib and IPA analysis. Illustration of the inhibited FOXM1 (b) and activated TP53 (c) networks is shown. Prediction legend indicated the relationship between different molecules in the network. (d) Bar graph plot depicting the top 10 activated and top 10 inhibited upstream networks based on differentially expressed genes in MDA-MB-231 cells treated with PTC-209 and IPA analysis.

Article Snippet: PTC-209 and palbociclib HCL small molecule inhibitors were purchased from Selleckchem (Houston, TX, USA).

Techniques:

Journal: Scientific Reports

Article Title: Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo

doi: 10.1038/s41598-019-50140-0

Figure Lengend Snippet: Combination of PTC-209 and palbociclib inhibited MDA-MB-231 tumor formation in vivo . ( a) MDA-MB-231 were treated with PTC-209, palbociclib, or combination of both and were implanted subcutaneously in nude mice as described in materials and methods. Data are presented as mean (tumor diameter) ± S.E.M., n = 2. ROC area under the curve was used to compare different growth curves. (b) Representative images of tumors excised from each treatment group at the end of the experiment. (c , d ) Representative histopathological examination of MDA-MB-231 xenograft tumors from each treatment group compared to vehicle-control treatment group. FFPE sections were stained with haematoxylin and eosin stain (c) or vimentin (d) . (Bar = 200 μm, panel c and d (upper); Bar = 100 μm, panel d (lower)). Black arrow indicates invading cells, square and rectangle indicates necrotic and dead cells and white arrow indicates mitotic events.

Article Snippet: PTC-209 and palbociclib HCL small molecule inhibitors were purchased from Selleckchem (Houston, TX, USA).

Techniques: In Vivo, Control, Staining, H&E Stain

Journal: Cell chemical biology

Article Title: Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1

doi: 10.1016/j.chembiol.2019.03.012

Figure Lengend Snippet: A, Immunopurified Flag-CDK4/myc-Cyclin D complexes phosphorylate recombinant 4E-BP1 and Rb. ***In vitro kinase assay using recombinant Rb as a substrate was run separately. Full Western blots for Rb shown in Figure S3A. B, Palbociclib (50 μM–500 pM) inhibits in vitro phosphorylation of 4E-BP1 by CDK4-cyclin D3. C, In vitro kinase assay using WT, S65A and S101A 4E-BP1 phosphorylated by CDK4-cyclin D3 complexes. D, MS analysis of in vitro phosphorylated 4E-BP1 by cyclin D3/CDK4. Phosphosites identified by manual curation of MS/MS assignments from ATP-treated sample are shown in red. No suitable phospho-peptides were identified in the no ATP control sample. Gold asterisk = canonical mTORC1 sites, Blue asterisk = non-canonical phosphorylation sites. E, HEK293T cells were transfected with indicated 3xFLAG-4EBP1 constructs. F, S101 (red) and the CDK4 recognition motif (blue) are highly conserved across mammals.

Article Snippet: Small molecule reagents Palbociclib Isethionate (Selleckchem) was dissolved in water to 10 mM.

Techniques: Recombinant, In Vitro, Kinase Assay, Western Blot, Tandem Mass Spectroscopy, Transfection, Construct

Journal: Cell chemical biology

Article Title: Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1

doi: 10.1016/j.chembiol.2019.03.012

Figure Lengend Snippet: A, Western blots of 3xFLAG-CDK4 and/or myc-D-cyclin transfected HEK293T cells stimulated and treated with rapamycin or DMSO following serum deprivation. B, Cells were deprived of serum, followed by stimulation with media containing serum and insulin with or without rapamycin and/or palbociclib. C, Colony formation of MCF-7, MDA-MB-468 and MDA-MB-231 cell lines treated with rapamycin and/or palbociclib. D, Quantification of cell density from (c). Signal intensity is normalized to the no treatment control. Error bars indicate standard deviation for 3 biological replicates. E, Western blot of MCF-7, MDA-MB-468 and MDA-MB-231 cell lines treated with rapamycin and/or palbociclib. Vertical bars separate samples run on separate blots or different exposures. F, Cap-dependent dual luciferase assay of cells treated with rapamycin, palbociclib and/or INK128. Normalized Renilla luciferase is shown relative to the no-treatment control for each cell line. Data represented as mean +/− standard deviation (n=4), and are representative of 3 independent replicates. *P<0.05, **P<0.01 ***P<0.001.

Article Snippet: Small molecule reagents Palbociclib Isethionate (Selleckchem) was dissolved in water to 10 mM.

Techniques: Western Blot, Transfection, Standard Deviation, Luciferase

Journal: Cell chemical biology

Article Title: Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1

doi: 10.1016/j.chembiol.2019.03.012

Figure Lengend Snippet: A, Western blot of c-Myc expression in MCF-7, MDA-MB-468, and MDA-MB-231 cell lines treated with rapamycin and/or palbociclib. B, Relative RNA expression of transcripts from cells treated as in (a). Expression is normalized to the no treatment control sample for each cell line. UBB was used as an internal control for each gene. Error bars standard deviation (n=3). Data is representative of 2 independent replicates. C, Expression of c-Myc in MDA-MB-468 and MCF-7 cell lines stably expressing doxycycline inducible FLAG-4E-BP1 mutants. D, Western blot of c-Myc expression in 4E-BP1 knockout or control cells treated as in A. Vertical bars separate images obtained from separate exposures. E, Western blot of cyclin D3 expression in 4E-BP1 knockout or control cells treated +/− palbociclib. F, Cap-dependent dual luciferase assay of cells treated with rapamycin and/or palbociclib. Data represented as mean +/− SEM (n=3) after normalization to no treatment control for the appropriate cell line. *P<0.05, **P<0.01.

Article Snippet: Small molecule reagents Palbociclib Isethionate (Selleckchem) was dissolved in water to 10 mM.

Techniques: Western Blot, Expressing, RNA Expression, Standard Deviation, Stable Transfection, Knock-Out, Luciferase

Journal: Cell chemical biology

Article Title: Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1

doi: 10.1016/j.chembiol.2019.03.012

Figure Lengend Snippet: A, Proliferation of MCF-7 cells +/− 4E-BP1 knockout treated with rapamycin and/or palbociclib assessed by CellTiter Glow assay. Treated samples are shown relative to no treatment control for that cell line. Bars represent mean +/− standard deviation (n=3) ***P<0.001, ****P<0.0001. B, List of proteins/antibodies with the highest correlation to survival in the BRCA dataset from TCPA. Shown are all antibodies with a Log-Rank P value of <0.01. P-values were used as reported by TCPA. C, Kaplain-Meyer curves showing the total survival time for BRCA patients from the TCPA dataset with high (red) and low (blue) p4E-BP1(S65/101). D, Histogram showing the Pearson Coefficient for pairwise regression between p4E-BP1 (S65/101) and every other antibody in the dataset.

Article Snippet: Small molecule reagents Palbociclib Isethionate (Selleckchem) was dissolved in water to 10 mM.

Techniques: Knock-Out, Standard Deviation

Journal: bioRxiv

Article Title: The kinase inhibitor Palbociclib is a potent and specific RNA-binding molecule

doi: 10.1101/2022.01.20.477126

Figure Lengend Snippet: a) Secondary structure of the HIV1-TAR RNA. b) The Parke-Davis group reported compounds which bind to the loop region of HIV-TAR (top). The amino pyrimidine core structure of the Parke-Davis fragment is found in several FDA-approved drugs, including Palbociclib (second from top) and Ribociclib; the third structure from the top is RBT-550 from the Ribotargets project , while the compound at the bottom is ‘Reverse Palbociclib’, as described in the main text. C) The three-dimensional structure of the HIV-TAR RNA in the presence of peptide JB-181, showing that the UCU bulge and apical loop are juxtaposed to each other, creating a new binding pocket that is absent in free TAR .

Article Snippet: Commercially available small molecule compounds (Palbociclib, Abemaciclib and Ribociclib) were purchased from Selleckchem, while ‘Reverse Palbociclib’ was synthesized in house as follows (Supplementary Fig. 8).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: The kinase inhibitor Palbociclib is a potent and specific RNA-binding molecule

doi: 10.1101/2022.01.20.477126

Figure Lengend Snippet: a) Structure of Palbociclib; b) Palbociclib peak intensities are plotted as a function of RNA concentration, with fits obtained using (see methods and reference 31) Spectra were normalized to the non-binding internal reference standard sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSA; 9 protons) and intensities were used to generate a binding isotherm, from which approximate binding constants were generated by curve fitting (◊ HIV TAR, • Pre-miR-21). c, d) 1H NMR titration series. 100 μM Palbociclib was titrated with HIV TAR in 10 μL steps of 500 μM RNA stock solution (0-5 μM) (c) and with pre-miR-21 RNA (0-5 μM) (d). These spectra were collected at 800Mhz and 37 °C, in 50 mM d9 deuterated bis-tris buffer at pH 6.5, containing 200 mM NaCl, 50 mM KCl and 4 mM MgCl2.

Article Snippet: Commercially available small molecule compounds (Palbociclib, Abemaciclib and Ribociclib) were purchased from Selleckchem, while ‘Reverse Palbociclib’ was synthesized in house as follows (Supplementary Fig. 8).

Techniques: Concentration Assay, Binding Assay, Generated, Titration

Journal: bioRxiv

Article Title: The kinase inhibitor Palbociclib is a potent and specific RNA-binding molecule

doi: 10.1101/2022.01.20.477126

Figure Lengend Snippet: (Top) 2-amino-purine (2-AP) HIV TAR binding assays and (Bottom) 1D imino-region NMR titrations for Palbociclib, Ribociclib and Abemaciclib. For Palbociclib, the 2-AP assay shows a rapid initial increase in fluorescence signal, indicating an increase in base stacking for 2-AP upon binding, as has been observed for other ligands , . This increase is followed by a decrease in the fluorescence signal at higher concentrations, as previously observed for aminoglycosides and Tat protein . This biphasic binding curve suggests the presence of two sites, a high affinity site with apparent KD of 104±80 nM (under low ionic conditions; 50 mM bis-Tris buffer) and a low affinity site with an apparent Kd >700 nM. In the NMR titrations, Palbociclib binding occurs in the slow exchange regime, consistent with nM affinity, while binding to Ribociclib is in the intermediate-slow regime, with behavior similar to Palbociclib but also significant line broadening. Abemaciclib binds in the intermediate regime, leading to exchange-broadened spectra, consistent with low μM/high nM affinity. The Palbociclib and Ribociclib titrations saturate at a 1:1 RNA-small molecule ratio; the top spectrum in each panel was recorded in the presence of excess small molecule and no further changes were observed.

Article Snippet: Commercially available small molecule compounds (Palbociclib, Abemaciclib and Ribociclib) were purchased from Selleckchem, while ‘Reverse Palbociclib’ was synthesized in house as follows (Supplementary Fig. 8).

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: The kinase inhibitor Palbociclib is a potent and specific RNA-binding molecule

doi: 10.1101/2022.01.20.477126

Figure Lengend Snippet: Binding of Palbociclib to the two RNAs occurs with very different affinity, as reflected in the NMR spectra. TOCSY spectra were collected at 800MHz under high salt buffer conditions (200 mM NaCl, 50 mM KCl and 4 mM MgCl2) at 37C. 250 μM RNA (black) was titrated with Palbociclib (red) until saturation was reached (as established from the absence of further changes in the spectra) and changes in chemical shifts were recorded. a) HIV TAR shows dramatic chemical shift changes and ‘slow exchange’ behavior between bound and free forms, with regular peak shapes for most signals, indicative of high affinity and specific binding, whereas b) pre-miR-21 shows much smaller chemical shift changes and irregular peak shapes, indicative of weaker affinity and a dynamic, poorly defined interaction site. These data support the conclusion that Palbociclib binds potently to HIV TAR but much more weakly to pre-miR-21, i.e. that the interaction is specific.

Article Snippet: Commercially available small molecule compounds (Palbociclib, Abemaciclib and Ribociclib) were purchased from Selleckchem, while ‘Reverse Palbociclib’ was synthesized in house as follows (Supplementary Fig. 8).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: The kinase inhibitor Palbociclib is a potent and specific RNA-binding molecule

doi: 10.1101/2022.01.20.477126

Figure Lengend Snippet: a) 0.50 nM of 5’ end P labeled HIV TAR were incubated with 0-90 nM of the preformed PTEFb/AFF4/Tat complex (by serial dilution). b) Palbociclib (10 nM) was pre-incubated with TAR RNA prior to binding (0-90 nM of the PTEFb/AFF4/Tat complex by serial dilution). c) Both assays were repeated in duplicate, as shown, and resolved on 6% native acrylamide gels; bands were quantified with imageJ and plotted vs concentration of the SEC complex. The binding affinity (KD) was calculated by curve fitting to be 0.34 ±0.1 nM for free TAR RNA, but it increased to 35.6 ±10 nM when 10.0 nM Palbociclib were preincubated with the RNA prior to complex formation.

Article Snippet: Commercially available small molecule compounds (Palbociclib, Abemaciclib and Ribociclib) were purchased from Selleckchem, while ‘Reverse Palbociclib’ was synthesized in house as follows (Supplementary Fig. 8).

Techniques: Labeling, Incubation, Serial Dilution, Binding Assay, Concentration Assay

Journal: Advanced Science

Article Title: VEGF‐FGF Signaling Activates Quiescent CD63 + Liver Stem Cells to Proliferate and Differentiate

doi: 10.1002/advs.202308711

Figure Lengend Snippet: VEGF and FGF signaling regulates proliferation and differentiation of QLSCs. A) Schematic diagram of experiments after silencing expression of Vegfr2 (Kdr) and Fgfr1. B,C) Western blotting and quantifications of liver lysates from each experimental group. FGFR1 or VEGFR2 protein pression was compared with GAPDH. Results are represented as mean ± SEM. n = 4. shR con, shRNA AAV control group. shR F1, shRNA Fgfr1 AAV group. shR V2, shRNA Vegfr2 AAV group. shR F1+V2, shRNA Fgfr1 and Vegfr2 AAV group. Con, control. D,E) Immunostaining for CD63, CD56, KRT19, VEGFR2 and FGFR1 in CD63 + CD56 − cells or liver section, isolated from shRNA (Vegfr2‐Fgfr1) AAV mice or control mice. Scale bar, 20 µm (D) or 100 µm (E). Nuclei were counterstained DAPI. F) Isolated QLSCs stained with Vybrant CFDA SE were imaged by fluorescence microscope. Day 1, up panels (1–8) showed cells cultured for 1 day. Day 21, bottom panels (1′‐8′ corresponding to 1–8) showed cells cultured for 3 weeks. F1 KD, Fgfr1 knockdown. V2 KD, Vegfr2 knockdown. V2+F1 KD, Vegfr2 and Fgfr1 both knockdown. Con, control AAV group. bF+VA, addition of bFGF and VEGF‐A in the culture medium. Neutral, neutralization of bFGF and VEGF‐A by antibodies (Mouse VEGF164 Antibody, 150 ng mL −1 and FGF basic/FGF2/bFGF Antibody, 200 ng mL −1 ). Inhibitors, addition small molecule inhibitors of VEGFR2 and FGFR1 (PD166866, 10 µ m and Anlotinib, 10 µ m ). iWithdrawal, withdrawal of chemical inhibitors. G) Fluorescence intensity of each group read by Microplate Reader. Results were showed as value ratio (D21/D1). Results are represented as mean ± SEM. n = 3. H–J) Proliferation and differentiation ability of CD63 + cells influenced by VEGF‐FGF signaling in vivo. H) Schematic diagram of AAV injection, injury process and interfering strategy. I) Representative images of immunostaining for CD63, EdU, HNF4A and GFP in the liver. Panel 1–4, EdU staining showed proliferation CD63 + cells. Panel 1′‐4′, GFP marked differentiated hepatocytes from CD63 lineage‐tracing mice liver. VA+bF Neural, neutralization of bFGF and VEGF‐A by antibodies. VEGF‐A+bFGF, VEGF‐A and bFGF injected group. Scale bar, 100 µm. Nuclei were counterstained DAPI. J) Calculation of EdU + CD63 + /total CD63 + cells (left) and GFP + hepatocytes/total hepatocytes (right) in each group. Data presented as mean ± SEM. n = 3. **** p < 0.0001.

Article Snippet: For in vitro, small molecules PD‐166866 and Anlotinib (Selleck), or growth factors VEGF‐A and bFGF, or neutral antibodies VEGF164 Antibody and bFGF Antibody, were dissolved and added in the medium.

Techniques: Expressing, Western Blot, shRNA, Control, Immunostaining, Isolation, Staining, Fluorescence, Microscopy, Cell Culture, Knockdown, Neutralization, In Vivo, Injection