Journal: The Febs Journal

Article Title: The m6A demethylase FTO promotes C/ EBPβ ‐ LIP translation to perform oncogenic functions in breast cancer cells

doi: 10.1111/febs.70033

Figure Lengend Snippet: FTO knockdown has a limited effect on mRNA translation. (A) Fold change in translation efficiency upon FTO knockdown in MDA‐MB‐231 cells is displayed, color coded by FDR. Significantly regulated genes above threshold are indicated by gene symbol. (B) RT‐qPCR analysis of DICER mRNA expression in MDA‐MB‐231 cells with FTO knockdown (shFTO1 or shFTO2) versus shRNA‐scrambled (scr) control. Significance was determined by one‐way ANOVA with Dunnett's post hoc test (mean ± SEM, n = 3 biological replicates). (C) Immunoblots showing no change in DICER protein expression upon knockdown of FTO (shFTO1 or shFTO2) in MDA‐MB‐231 versus scrambled control (SCR). β‐Actin was used as loading control. Quantification is shown on the right. Significance was determined by one‐way ANOVA with Dunnett's post hoc test (mean ± SEM, n = 3 biological replicates). (D) RT‐qPCR analysis of COL1A1 and SMAD6 mRNA expression in MDA‐MB‐231 upon FTO knockdown (shFTO1 or shFTO2) versus shRNA‐scrambled (scr) control. Significance was determined by one‐way ANOVA with Dunnett's post hoc test (mean ± SD, n = 3 biological replicates). (E) Immunoblot analysis of COL1A1, SMAD6, and DICER protein expression in MDA‐MB‐231 upon FTO knockdown (shFTO1 or shFTO2) versus shRNA‐scrambled (scr) control, and β‐Actin as loading control. 5 days and 20 days refer to days in cell culture after transduction with shRNA retroviral vector. (F) Subsequence abundance analysis of codons at the +15 position relative to the ribosome protected fragment (RPF) 5′‐end. All transcripts in the coding frame are considered for this analysis. No significant enrichment or depletion of codons can be seen, indicating no obvious shortages of amino acids causing ribosome stalls/halts.

Article Snippet: The following primary antibodies were used for detection: FTO: NB110‐60935, Novus Biologicals, Abingdon, UK (1:1666); β‐Actin: 691001, MP Bio, Heerlen, The Netherlands (1:5000); C/EBPβ: ab32358, Abcam, Cambridge, UK (1:1000); WTAP: #56501 (1:1000), Cell Signaling Technology, Leiden, The Netherlands; COL1A1: #72026 (1:1000), Cell Signaling Technology, Leiden, The Netherlands; SMAD6: NB100‐56440, Novus Biologicals, Abingdon, UK (1:1000), FLAG‐M2: F‐3165, Sigma Aldrich, Merck, Dordrecht, The Netherlands (1:1000).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot, Cell Culture, Transduction, Retroviral, Plasmid Preparation

Journal: Life Science Alliance

Article Title: Tissue-specific I-Smad mechanisms revealed by structure–function analysis in Drosophila

doi: 10.26508/lsa.202503445

Figure Lengend Snippet: (A, B) AlphaFold3 structural predictions of MH1 domains. The β-hairpin region is shown in yellow. Remaining MH1 is shown in blue. (A) Smad3 MH1 domain showing DNA-binding region and predicted interaction. (B) Dad, Smad6, and Smad7 MH1 domains. (C) Representative wings from females expressing indicated construct in wing primordium. The scale bar represents 1 mm. (D, E) Transgenic control ( TwitGFP , OK371-Gal4/+ ) compared with OK371-Gal4 –driven overexpression of UAS-Dad , UAS-Smad6 , UAS-Smad7L , and UAS-Smad7S . (D) Motor neurons in ventral nerve cord triple-labeled for Elav (blue), TwitGFP (green), and pMad (red). The scale bar represents 20 microns. (E) Quantification of pMad and TwitGFP intensity normalized to the genetic background control. Bars represent the mean ± SEM. Each dot represents mean data from motor neurons of one CNS. Statistical comparisons were performed using Dunn’s test with the Bonferroni correction. **** P < 0.0001, ** P < 0.01. Number of brains analyzed for pMad in the order shown in the graph: 52, 24, 25, 20, and 28. Number of brains analyzed for TwitGFP in the order shown in the graph: 53, 27, 22, 19, and 26.

Article Snippet: CS2 Smad6 , N/A , CS2 Smad6 was a gift from Joan Massague (plasmid #14960; https://n2t.net/addgene:14960 ; RRID:Addgene_14960; Addgene) ( ) , Source of Smad6 CDS.

Techniques: Binding Assay, Expressing, Construct, Transgenic Assay, Control, Over Expression, Labeling

Journal: Endocrinology

Article Title: Inhibitor of Differentiation (Id) genes are expressed in the steroidogenic cells of the ovine ovary and are differentially regulated by members of the Transforming Growth Factor (TGF)-? family

doi: 10.1210/en.2009-0914

Figure Lengend Snippet: List of primary, secondary and tertiary antibodies used for immunohistochemistry (IH) and immunofluorescence (IF). Secondary antibody; 1) Biotinylated Goat anti-Rabbit IgG (Dako, Glostrup, Denmark) 1:500, 2) Peroxidase Goat anti-Rabbit (Dako) 1:200. Tertiary antibody; A) Streptavidin Alexafluor 546 (s-11225 Invitrogen, Paisley, UK) 1:200, B) Tyramide Fluorescein (Perkin Elmer LAS, Inc. MA, USA) 1:50.

Article Snippet: Smad 6 , Polyclonal Rabbit (IMG-555), Imgenex Corp, Cambridge BioScience, Cambs, UK , IH 1:100 IF 1:600 , 2 , B.

Techniques: Immunohistochemistry, Immunofluorescence