Journal: bioRxiv

Article Title: m 6 A Demethylase FTO Stabilizes LINK-A to Exert Oncogenic Roles via MCM3-Mediated Cell Cycle Progression and HIF-1α Activation

doi: 10.1101/2023.01.28.526069

Figure Lengend Snippet: (A) A list of LINK-A-associated proteins identified by MS following RNA pulldown assays in KYSE450 and YES2 cells. (B) Western blot analysis of MCM3 expression following RNA pulldown assays in the indicated cells. (C) RT‒qPCR analysis of LINK-A and U1 expression following RIP assays in the indicated cells. The data are presented as the mean ± s.d. values; unpaired Student’s t test, ***p < 0.001; n = 3. (D and E) Western blot analysis of MCM3 and phosphorylated MCM3 in the indicated cells. (F and G) RT‒qPCR analysis of LINK-A expression (F) and Western blot analysis of MCM3 and phosphorylated MCM3 (G) after exogenous expression of increasing amounts of LINK-A in KYSE410 cells. (H) Western blot analysis of CDK1 expression following RNA pulldown assay in the indicated cells. (I and J) Western blot analysis of IP results in the indicated cells. (K and L) Representative images of the merged PLA and nuclear (DAPI) channels and quantitative analysis of PLA spots in the indicated cells. The scale bar represents 10 μm. The data are presented as the mean ± s.e.m. values; unpaired Student’s t test, ** p < 0.01, ***p < 0.001; n = 3.

Article Snippet: The following antibodies were used for Western blot analysis: anti-MCM3 (#4012; Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-CDK1 (#9116; Cell Signaling Technology; 1:1000), anti-HIF-1α (#36169; Cell Signaling Technology; 1:1000), anti-phospho-MCM3 (Ser112) (#12686; Cell Signaling Technology; 1:1000), anti-IGF2BP2 (ab124930; Abcam, Cambridge, MA, USA; 1:1000), anti-IGF2BP1 (100999; Abcam; 1:1000), anti-IGF2BP3 (ab177477; Abcam; 1:1000), anti-FTO (712913; Thermo Scientific; 1:1000), anti-MCM2 (A1056; ABclonal, Wuhan, Hubei, China; 1:1000), anti-MCM4 (A13513; ABclonal; 1:1000), anti-MCM5 (A5556; ABclonal; 1:2000), anti-MCM6 (A1955; ABclonal; 1:1000), anti-MCM7 (A1138; ABclonal; 1:1000), anti-H3 (ab1791; Abcam; 1:1000), anti-Lamin B1 (A1910; ABclonal; 1:2000), and anti-β-actin (A5316; Sigma–Aldrich; 1:4000).

Techniques: Western Blot, Expressing

Journal: Discover. Oncology

Article Title: Lenvatinib inhibits intrahepatic cholangiocarcinoma via Gadd45a-mediated cell cycle arrest

doi: 10.1007/s12672-023-00631-4

Figure Lengend Snippet: Gadd45a is a key factor by which lenvatinib induces cell cycle arrest in ICC. a Gadd45a protein and mRNA silencing in ICC cells was confirmed by immunoblotting analysis and quantitative RT-PCR, respectively; b and c the proportion of cells in each phase of the cell cycle and the viability of cells in Gadd45a-silenced and Gadd45a-scramble cell lines following lenvatinib treatment (all statistics are based on comparison with the control group); d immunoblotting analysis of Gadd45a, CDK1 and CDK2 levels in ICC cells with Gadd45a silencing or scramble following lenvatinib treatment. The grouping of blots cropped from different parts of the same gel or from different gels, fields, or exposures is divided with white space. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The proteins were then transferred to a polyvinylidene fluoride membrane, which was then incubated with diluted primary antibodies (Abcam Inc., Cambridge, UK), including rabbit antibodies against Actin (1:5000, Proteintech, 23660-1-AP), CDK1 (1:1000, Cell Signaling Technology, 9116), CDK2 (1:1000, Cell Signaling Technology, 18048), CyclinB1 (1:1000, Proteintech, 55004-1-AP), Cyclin E (1:1000, Proteintech, 11554-1-AP), p21 (1:5000, Abcam Inc., ab109520) and Gadd45a (1:1000, Abcam Inc., ab180768), overnight at 4 °C.

Techniques: Western Blot, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Combination of EphA2- and Wee1-Targeted Therapies in Endometrial Cancer

doi: 10.3390/ijms24043915

Figure Lengend Snippet: High–throughput screen for synergistic partners for EphA2–targeted therapy and verification of Wee1 as a target. ( A ) Top 10 hits from the chemical screen. ( B ) Effect of EphA2 silencing on Wee1 activity, as seen by phosphorylation of cdc2, in Hec1A cells. ( C ) Dose–response matrix for combinatorial analysis of the EphA2 inhibitor ALW and Wee1 inhibitor MK1775. ( D ) Difference from predicted Bliss independence surface model for the combination of ALW and MK1775. ( E ) Visualization of the calculated 2D synergy maps for the MTT cell viability assay from the SynergyFinder Bliss independence model combinatorial analysis. Red regions represent synergy, and green regions represent antagonism. ( F ) Plots showing fraction of cells affected (Fa) and combination index (CI) values for ALW and MK1775. The white circles representing the CI values of the ALW:MK1775 combination (1 µM:0.5 µM and 1 µM:0.75 µM concentrations, respectively) were less than one, indicating synergy.

Article Snippet: The following antibodies were used: anti-EphA2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated (phospho) cdc2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-cdc2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), Cell Cycle and Apoptosis WB Cocktail (pCdk/pHH3/Actin/cleaved PARP) (1:250 dilution; Abcam, Cambridge, UK); Apoptosis and DNA damage WB Cocktail (pH2A.X/GAPDH/cleaved PARP) (1:250 dilution; Abcam, Cambridge, UK); anti-phospho S6 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-S6 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-AKT (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA); anti-pAKT (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA); anti-cleaved caspase 3 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA); anti-P62 (1:3000; BD Biosciences, Franklin Lakes, NJ, USA); anti-GAPDH (1:5000; Thermo Fisher Scientific, Waltham, MA, USA), anti-LC3B (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-alpha-tubulin (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), and antibeta-actin (1:3000; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: High Throughput Screening Assay, Activity Assay, Viability Assay

Journal: International Journal of Molecular Sciences

Article Title: Combination of EphA2- and Wee1-Targeted Therapies in Endometrial Cancer

doi: 10.3390/ijms24043915

Figure Lengend Snippet: High–throughput screen for synergistic partners for EphA2–targeted therapy and verification of Wee1 as a target. ( A ) Top 10 hits from the chemical screen. ( B ) Effect of EphA2 silencing on Wee1 activity, as seen by phosphorylation of cdc2, in Hec1A cells. ( C ) Dose–response matrix for combinatorial analysis of the EphA2 inhibitor ALW and Wee1 inhibitor MK1775. ( D ) Difference from predicted Bliss independence surface model for the combination of ALW and MK1775. ( E ) Visualization of the calculated 2D synergy maps for the MTT cell viability assay from the SynergyFinder Bliss independence model combinatorial analysis. Red regions represent synergy, and green regions represent antagonism. ( F ) Plots showing fraction of cells affected (Fa) and combination index (CI) values for ALW and MK1775. The white circles representing the CI values of the ALW:MK1775 combination (1 µM:0.5 µM and 1 µM:0.75 µM concentrations, respectively) were less than one, indicating synergy.

Article Snippet: The following antibodies were used: anti-EphA2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated (phospho) cdc2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-cdc2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), Cell Cycle and Apoptosis WB Cocktail (pCdk/pHH3/Actin/cleaved PARP) (1:250 dilution; Abcam, Cambridge, UK); Apoptosis and DNA damage WB Cocktail (pH2A.X/GAPDH/cleaved PARP) (1:250 dilution; Abcam, Cambridge, UK); anti-phospho S6 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-S6 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-AKT (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA); anti-pAKT (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA); anti-cleaved caspase 3 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA); anti-P62 (1:3000; BD Biosciences, Franklin Lakes, NJ, USA); anti-GAPDH (1:5000; Thermo Fisher Scientific, Waltham, MA, USA), anti-LC3B (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-alpha-tubulin (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), and antibeta-actin (1:3000; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: High Throughput Screening Assay, Activity Assay, Viability Assay