Journal: Nutrients

Article Title: Anticancer and Antioxidant Effects of Bioactive Peptides from Black Soldier Fly Larvae ( Hermetia illucens ).

doi: 10.3390/nu17040645

Figure Lengend Snippet: Figure 6. Overview of microarray results and protein expression analysis in ASBP-AH3-treated COLO205 cells: (A) the compilation of upregulated (z-score > 2) and downregulated (z-score < −2) pathways identified from the Diseases and Bio Functions dataset following LC50 treatment of COLO205 cancer cells with ASBP-AH3; (B) the heatmap of log2-transformed signal values for inactivated genes from the same dataset; (C) the STRING protein–protein interaction network of inactivated genes from the Diseases and Bio Functions dataset; (D) Western blot analysis; and (E–G) expression ratios of SKP2, p21, and cyclin D1 proteins in the ASBP-AH3-treated group com- pared to control. Data are presented as mean ± SD (n = 3), with differences between groups determined using Student’s t-test (* p < 0.05, ** p < 0.01).

Article Snippet: Primary antibodies included anti-Skp2 (1:1000), anti-p21 Waf1/Cip1 (1:1000), anti-Cyclin D1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (1:50,000, Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Microarray, Expressing, Transformation Assay, Western Blot, Control

Journal: Cell Cycle

Article Title: A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

doi: 10.4161/cc.23515

Figure Lengend Snippet: Figure 1. Identification of senescence-inducing kinase siRNAs. ( A ) Schematic overview of the high-content senescence screen. ( B ) Scatterplot analysis of data from the kinome HCS screen in p53+ cells. Cells were stained for Ki67, p53, p21 CIP1 and Hoechst at day 3, and multiparametric analysis was performed to determine siRNAs displaying decreased proliferation (< 0.5 StDev Ki67%) and an increased nuclear size (> 2.0 StDev Hoechst mean area). High, medium and low cell number indicators are assigned as the third parameter. ( C ) Senescence scores per gene are the sum of the senescence scores of its siRNA pool and the four individual siRNAs, omitting siRNAs conferring less than 50% specific gene knockdown. Scoring details are depicted in . References to studies establishing bona fide tumor suppressor functions for human ( Hs ) or mouse ( Mm ) genes are indicated.

Article Snippet: Primary antibodies were rabbit anti-p53 (Cell Signaling 928; 1:1500), goat anti-p21 (R&D Systems AF1047; 1:20.000) and mouse-anti-Ki67 (BD PharMingen 55600; 1:1500).

Techniques: Staining, Knockdown

Journal: Cell Cycle

Article Title: A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

doi: 10.4161/cc.23515

Figure Lengend Snippet: Figure 2. Delineation of senescence signatures. ( A ) hTERT-RPE1 p53 shRNA cells were transfected with kinome siRNAs in the absence of doxycycline and immunostained for p53 and p21 CIP1 following 72 h culture. Percentages of cells expressing p53, p21 CIP1 above threshold levels were calculated, and averages from four 384 wells are shown. ( B ) hTERT-RPE1 p53 shRNA cells were transfected with kinome siRNAs in the absence or presence of doxycycline and immunostained for Ki67. Percentages of cells expressing Ki67 protein above threshold levels were calculated, and averages from four 384 wells are shown. * indicates p ≤ 0,05 and ** indicates p ≤ 0,01. ( C ) Senescence scores per gene are the sum of the senescence scores of the siRNA pool plus four individual siRNAs, omitting siRNAs conferring less than 50% knockdown. Detailed scoring information is depicted in . ( D ) Quantitative p16 INK4A mRNA expression analyses during senescence induction. hTERT-RPE1 p53 shRNA cells were transfected with pooled siRNAs in the absence of doxycycline. RNA expression was quantitated using TaqMan analyses after 3 days of transfection. ( E ) Schematic model summarizing kinome screen data, using data depicted in ( A ) and ( D ). In an incipient tumor, modeled by hTERT-RPE1 cells, loss of selected tumor suppressors activates p53- and/or p16 INK4A -dependent senescence, and overt DNA damage. In a premalignant tumor, senescence may serve as a cell-intrinsic tumor suppressor mechanism to subvert oncogenic transformation. Loss of p16 INK4A and/or p53 promotes malignancy.

Article Snippet: Primary antibodies were rabbit anti-p53 (Cell Signaling 928; 1:1500), goat anti-p21 (R&D Systems AF1047; 1:20.000) and mouse-anti-Ki67 (BD PharMingen 55600; 1:1500).

Techniques: shRNA, Transfection, Expressing, Knockdown, RNA Expression, Transformation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: ATE1 promotes breast cancer progression via arginylation-dependent regulation of MAPK-MYC signaling

doi: 10.1186/s12964-025-02376-9

Figure Lengend Snippet: The role of ATE1 in MYC stability. a T-47D and MDA-MB-157 cells with ATE1 knockdown (shATE1#69) were synchronized using the double thymidine (2mM) block method. Following release at the indicated time points, cell lysates were collected. The expression levels of endogenous MYC, p-MYC (S-62), p-ERK1/2 (T202/Y204), total ERK1/2, p-AKT (S-473), p-AKT (T-308), and total AKT were analyzed by western blot. b The effect of ATE1 depletion on the expression of MYC, p27, p21, and p16 in T-47D cells, as outlined in a , was assessed by western blot analysis. c T-47D cells were transfected with 20 nM siRNA (ATE1#1) against ATE1 or a scrambled negative control for 36 h. After cycloheximide (CHX, 10 µM) treatment, cells were harvested at 30-min intervals, lysed, and analyzed by western blot using a MYC antibody. d The intensities of MYC bands were measured using ImageJ software. The graph shows the percentage of MYC remaining relative to β-actin. Data are presented as mean ± SD from three independent experiments ( n = 3). Statistical significance was determined by two-tailed unpaired t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). For all western blot panels, band intensities were quantified, normalized to β-actin, and presented as mean ± SD from three independent biological replicates

Article Snippet: The following primary antibodies were used for immunoblotting: ATE1 (Santa Cruz Biotechnology #sc-271220), β-actin (Sigma #A1978), cyclin D (Santa Cruz Biotechnology #sc-8396), Cdk4 (Santa Cruz Biotechnology #sc-23896), Cdk6 (Santa Cruz Biotechnology #sc-7961), phospho-Rb (Ser795) (Cell Signaling Technology #9301), Rb (Santa Cruz Biotechnology #sc-50), cyclin E (Santa Cruz Biotechnology #sc-25303), cyclin A (Santa Cruz Biotechnology #sc-751), Cdk2 (Santa Cruz Biotechnology #sc-6248), cyclin B (Santa Cruz Biotechnology #sc-166152), c-Myc (D84C12) (Cell Signaling Technology #5605), c-Myc (9E10) (Santa Cruz Biotechnology #sc-40), p27 Kip1 (Cell Signaling Technology #3686), p21 Waf1/Cip1 (Cell Signaling Technology #2946), p16 (Santa Cruz Biotechnology #sc-81613), GAPDH (Santa Cruz Biotechnology #sc-47724), phospho-c-Myc (Ser62) (abcam #ab78318), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology #9106), p44/42 MAPK (Erk1/2) (Cell Signaling Technology #9102), phospho-Akt (Ser473) (Cell Signaling Technology #9271), phospho-Akt (Thr308) (Cell Signaling Technology #9275), Akt1 (Santa Cruz Biotechnology #sc-5298), PARP-1 (H-250) (Santa Cruz Biotechnology #sc-7150), Caspase-3 (Pro and Active) (IMGENEX #IMG-144 A), E-cadherin (Cell Signaling Technology #3195), and vimentin (Santa Cruz Biotechnology #sc-6260).

Techniques: Knockdown, Blocking Assay, Expressing, Western Blot, Transfection, Negative Control, Software, Two Tailed Test

Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a IB analysis of WCLs derived from HEK293T cells transfected with His-E4F1 and increasing doses of Flag-PA28γ. b Quantitative real-time PCR analysis of the indicated genes in HEK293T cells transfected with EV and increasing doses of Flag-PA28γ. Endo., endogenous. c HSC-3 cells stably expressing EV or Flag-PA28γ were treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of E4F1 levels relative to tubulin levels is shown. d , e HEK293T cells stably expressing EV or Flag-PA28γ were transfected with Myc-E4F1 truncations, and treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of Myc-E4F1 levels relative to tubulin levels is shown. f HEK293T cells transfected with the indicated plasmids were treated without or with 10 μM MG132 for 6 h before harvesting, and WCLs were collected for IB analysis. g HEK293T cells stably expressing shRNA against endogenous PA28γ were transfected with the indicated plasmids, and WCLs were collected for IB analysis. h Purified PA28γ, E4F1 and 20S proteasome in the absence or presence of 100 nM proteasome inhibitor epoxomicin (Epox) were incubated as indicated for 45 min, followed by IB analysis. i Purified PA28γ WT, PA28γ-T23A, E4F1, p21 (as a positive control for E4F1) and 20S proteasome were incubated as indicated for 45 min, followed by IB analysis. Data in ( b – e ) represent the mean ± SD of three biological replicates; statistical significance was assessed by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human E4F1 protein (H00001877-P01) and recombinant human p21/CDKN1A protein (NBP2-22976) were purchased from Novus Biologicals.

Techniques: Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, shRNA, Purification, Incubation, Positive Control