Journal: Molecules

Article Title: Anti-Invasive and Apoptotic Effect of Eupatilin on YD-10B Human Oral Squamous Carcinoma Cells

doi: 10.3390/molecules30244666

Figure Lengend Snippet: Cell cycle analysis of eupatilin on YD-10B cells. ( A ) Cell cycle distribution was analyzed by flow cytometry after treatment with various concentrations of eupatilin (0–50 μM) for 48 h. Histograms showed the proportion of cells in the G 0 /G 1 , S, and G 2 /M phases. The box in panel A indicates the cell cycle phase distribution (%). ( B ) Quantitative analysis was performed to determine the percentage of cells in each phase of the cell cycle after eupatilin treatment. ( C ) mRNA expression levels of Cyclin D1 and CDK2 were determined by RT-qPCR analysis. ( D ) Protein expression levels of Cyclin D1, CDK2, and p21 were examined by Western blot analysis. γ-Tubulin and GAPDH were used as loading controls. Relative band intensities were quantified using ImageJ (version 1.54g) software. Data represent the mean ± SD of three independent experiments (*; p < 0.05, **; p < 0.01, ***; p < 0.001 compared with untreated control).

Article Snippet: Cyclin D1 (#A19038; Abclonal, Wuhan, China), CDK2 (#2546; Cell Signaling Technology, Danvers, MA, USA), p21 (#2947; Cell Signaling Technology), Bax (#14796, Cell Signaling Technology), Bcl-2 (#15071; Cell Signaling Technology), caspase-9 (#A18676; Abclonal) (#9542; Cell Signaling Technology), caspase-3 (#9662; Cell Signaling Technology), cleaved caspase-3 (#9661; Cell Signaling Technology), PARP (#9542; Cell Signaling Technology), MMP-2 (#A6247; Abclonal), MMP-9 (#A0289, Abclonal), γ-Tubulin (#sc-7396; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAPDH (#AC033; Abclonal) were diluted 1:1000 and added and reacted at 4 °C for 12 h. Goat anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody (#7074P2; Cell Signaling Technology), Horse anti-mouse HRP-conjugated IgG secondary antibody (#7076P2; Cell Signaling Technology), and Donkey anti-goat HRP-conjugated IgG secondary antibody (#sc-2020; Santa Cruz Biotechnology) were diluted 1:5000 and reacted at room temperature for 1 h. After that, the chemiluminescence was measured using an enhanced chemiluminescence (ECL) kit (Amersham pharmacia Biotech Ltd., Amersham, UK).

Techniques: Cell Cycle Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Software, Control

Journal: The EMBO Journal

Article Title: Resistance to CDK7 inhibitors directed by acquired mutation of a conserved residue in cancer cells

doi: 10.1038/s44318-025-00554-6

Figure Lengend Snippet: ( A ) Mean IC 50 values from n = 3 independent experiments for 22Rv1 and 22Rv1-SamR cells. These results are summarised in Fig. . Error bars = SEM. ( B ) Densitometric quantification of immunoblots from n = 3 independent experiments in which 22Rv1 and 22Rv1-SamR cells were treated with the indicated concentrations of Samuraciclib or SY1365. Signal intensities are shown relative to the appropriate vehicle (0 nM) controls. One of the immunoblots used for the quantification is shown in Fig. . Error bars show SEM. Pairwise comparisons between treated groups and vehicle were performed using repeated measures of one-way ANOVA followed by Fisher’s LSD post hoc test (uncorrected). Asterisks indicate significance (* P < 0.05, ** P < 0.01). P value for P-Ser5, 22Rv1 cells: Veh vs Sam (100 nM) P = 0.0093; Veh vs Sam (1000 nM) P = 0.0232. 22Rv1-SamR cells: Veh vs SY1365 (5 nM) P = 0.0227; Veh vs SY1365 (50 nM) P = 0.0052. For P-CDK2, 22Rv1 cells: Veh vs Sam (100 nM) P = 0.0148; Veh vs Sam (1000 nM) P = 0.0017; Veh vs SY1365 (50 nM) P = 0.0268. 22Rv1-SamR cells: Veh vs Sam (1000 nM) P = 0.0027; Veh vs SY1365 (50 nM) P = 0.0102. For β-actin, 22Rv1-SamR cells: Veh vs sam (1000 nM) P = 0.0441. No asterisk indicates a non-significant difference ( P > 0.05). ( C ) RNA-seq was performed using six RNA samples prepared from 22Rv1 and 22Rv1-SamR cells. Shown are normalised read counts for CDK7 and its interacting partners cyclin H (CCNH) and MAT1 (MNAT1). Circles represent the normalised read counts for each of the six biological replicate RNA samples. ( D , E ) Genome browser snapshots (hg38) of RNA-seq data for 22Rv1 (SAMSEN) and 22Rv1-SamR (SAMRES) cells are shown. The vertical bars show positions of two known CDK7 SNPs (rs2972388 (exon 2) and rs34584424 (exon 10)), which are present in the Sam-sensitive and resistant cells at a ratio of 1:1, suggestive of the presence of 2 CDK7 alleles in 22Rv1 cells. Also evident is a single nucleotide difference (exon 5; c.289G>A) in the codon encoding aspartate 97 (p.Asp97Asn), which is seen only in 22Rv1-SamR cells. .

Article Snippet: Subsequently, the membranes were destained by washing in PBST (1× phosphate-buffered saline supplemented with 0.1% v/v Tween 20) and immunoblotting was performed using a rabbit anti-phospho-CDK2 (Thr160) primary antibody (Cell Signaling Technology, cat. #2561) diluted 1:1000.

Techniques: Western Blot, RNA Sequencing

Journal: The EMBO Journal

Article Title: Resistance to CDK7 inhibitors directed by acquired mutation of a conserved residue in cancer cells

doi: 10.1038/s44318-025-00554-6

Figure Lengend Snippet: ( A – D ) HEK293 cells were transfected with Nano-luc CDK7 fusion constructs to investigate binding of different CDK7i, using the NanoBRET Target Engagement assay for fluorescent tracer (K-10) displacement ( n = 3 independent experiments; error bars = SEM). Occupancy (%) represents the CDK7 fractional occupancy of CDK7 as a measure of tracer displacement with increasing concentration of CDK7i. ( E ) Western blot detection of T-loop phosphorylated CDK2 from three kinase reactions using recombinant CAK D97N (top) or CAK WT (bottom) and recombinant CDK2 as the substrate. Ponceau S staining was used to provide a loading control. Each membrane contains one control lane where the product of a 1-h reaction using the other CDK7 variant was detected to confirm successful blotting and detection and facilitate comparison of the results. ( F ) Quantification of the Western blot band intensities shown in ( E ), presented as the mean ± SD of N = 3 technical replicates. Significance levels were assessed using two-way ANOVA with correction for multiple comparisons (* P < 0.05; *** P < 0.001). .

Article Snippet: Subsequently, the membranes were destained by washing in PBST (1× phosphate-buffered saline supplemented with 0.1% v/v Tween 20) and immunoblotting was performed using a rabbit anti-phospho-CDK2 (Thr160) primary antibody (Cell Signaling Technology, cat. #2561) diluted 1:1000.

Techniques: Transfection, Construct, Binding Assay, Drug discovery, Concentration Assay, Western Blot, Recombinant, Staining, Control, Membrane, Variant Assay, Comparison

Journal: Biochimica et biophysica acta

Article Title: Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

doi: 10.1016/j.bbadis.2016.07.017

Figure Lengend Snippet: Fig. 4. Apoptosis and cell cycle arrest by miR-5582-5p occur through the downregulation of GAB1/SHC1 and CDK2, respectively. (A) miR-5582-5p suppresses activation of AKT and ERK downstream of growth factor signaling. HCT116 cells transfected with control or miR-5582-5p were serum starved for 24 h, and then stimulated with EGF (100 ng/mL). Cells were harvested at the indicated time points for Western blot analysis. (B) Combined knockdown of GAB1 and SHC1 induces apoptosis. Flow cytometric analyses were carried out 3 days after transfection of HCT116 cells with miR-5582-5p or siGAB1, siSHC1, or both. (C) Knockdown of GAB1 and SHC1 mimics the effect of miR-5582-5p in the regulation of apoptosis- related proteins downstream of growth factor signaling. HCT116 cells were transfected with miR-5582-5p or siRNA specific for GAB1 or SHC1 as indicated, and the expression of target proteins was analyzed 48 h after transfection. (D) Comparison of cell cycle arrest induction by transfection with siCDK2 and miR-5582-5p. HCT116 cells transfected with miR-5582-5p or siCDK2 were analyzed for cell cycle distribution 48 h after transfection. (E) Knockdown of CDK2 mimics the effect of miR-5582-5p on the expression of cell cycle-related proteins. HCT116 cells were transfected with miR-5582-5p or siCDK2, and the expression of target proteins was analyzed 48 h after transfection. (F) Confirmation of induction of miR-5582-5p expression by doxycycline treatment in the established Tet-5582 cell line. Total RNA was extracted from Tet-C or Tet-5582 cells at the indicated times after doxycycline (Dox) treatment (500 ng/mL), expression level of miR-5582-5p was analyzed by qRT-PCR. Error bars represent mean ± SEM. (G) Alteration in the expression of apoptosis-associated target proteins upon induced expression of miR-5582-5p. Cell lysates of Tet-C and Tet-5582 were prepared at the indicated times after doxycycline treatment, and analyzed for the expression of target proteins by Western blotting. (H) Induced expression of miR-5582-5p induces apoptosis. Apoptosis analyses were carried out 72 h after induction with doxycycline treatment. (I) Alteration in the expression of cell cycle-associated target proteins upon induced expression of miR-5582-5p. (J) Induced expression of miR-5582-5p induces cell cycle arrest.

Article Snippet: An inhibitor of miR-5582-5p was also synthesized by Genolution Pharmaceuticals as a 2′-O-methylmodified oligoribonucleotide single strand with a sequence of 5′- GCTATAACTTTAAGTGTGCCTA-3′. siRNA duplex oligonucleotides targetingGAB1, SHC1, and CDK2, and scrambled non-targeting negative control siRNAwere purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Transfection, Control, Western Blot, Knockdown, Expressing, Comparison, Quantitative RT-PCR

Journal: Biochimica et biophysica acta

Article Title: Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

doi: 10.1016/j.bbadis.2016.07.017

Figure Lengend Snippet: Fig. 5. Restoration of GAB1/SHC1 or CDK2 suppresses miR-5582-5p-induced apoptosis or cell cycle arrest, respectively. (A). Determination of GAB1 and SHC1 levels after ectopic expression. The effect of GAB1 and SHC1 overexpression in the regulation of miR-5582-5p induced apoptosis. HCT116 cells were co-transfected with miR-5582-5p in combination with Flag-GAB1 and Flag-SHC1 expression vectors as indicated. (B) Combined overexpression of GAB1 and SHC1 suppresses miR-5582-5p-induced apoptosis. Flow cytometric analyses were carried out 72 h after transfection of HCT116 cells as indicated. (C) Determination of CDK2 levels after ectopic expression. (D) Ectopic expression of CDK2 suppresses miR-5582- 5p-induced cell cycle arrest. Cell cycle distribution was analyzed by flow cytometry after transfection of HCT116 cells as indicated.

Article Snippet: An inhibitor of miR-5582-5p was also synthesized by Genolution Pharmaceuticals as a 2′-O-methylmodified oligoribonucleotide single strand with a sequence of 5′- GCTATAACTTTAAGTGTGCCTA-3′. siRNA duplex oligonucleotides targetingGAB1, SHC1, and CDK2, and scrambled non-targeting negative control siRNAwere purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Over Expression, Transfection, Cytometry

Journal: Biochimica et biophysica acta

Article Title: Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

doi: 10.1016/j.bbadis.2016.07.017

Figure Lengend Snippet: Fig. 7. miR-5582-5p is downregulated in CRC tissues and shows inverse correlation with GAB1, SHC1, and CDK2 expression. (A) Comparison of miR-5582-5p expression in tumor and normal tissues from CRC patients. Total RNA was extracted from 10 pairs of frozen tumor and adjacent normal tissues from CRC patients. Error bars represent mean ± SEM. (B) Comparison of target protein expression in tumor and normal tissues from CRC patients. Tissue lysates were prepared through homogenization in RIPA buffer from the same tissue samples used for the determination of miR-5582-5p expression shown in (A).

Article Snippet: An inhibitor of miR-5582-5p was also synthesized by Genolution Pharmaceuticals as a 2′-O-methylmodified oligoribonucleotide single strand with a sequence of 5′- GCTATAACTTTAAGTGTGCCTA-3′. siRNA duplex oligonucleotides targetingGAB1, SHC1, and CDK2, and scrambled non-targeting negative control siRNAwere purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Comparison, Homogenization