Journal: The FEBS journal

Article Title: FKBP25 regulates myoblast viability and migration and is differentially expressed in in vivo models of muscle adaptation.

doi: 10.1111/febs.16894

Figure Lengend Snippet: Fig. 2. FKBP25 protein is increased in quiescent myoblasts and reduced upon re-entry into the cell cycle. C2C12 myoblasts were suspended in a semi-solid medium (0.04%w/v methylcellulose/DMEM) for 48 to enter a state of cell cycle arrest. (A) Upon removal from suspension culture, cells were replated on a standard tissue culture substratum. It was observed that there were no morphological changes to myo- blasts or differentiated myotubes (Scale bar = 100 lm). (B) FKBP25 protein expression is indifferent in suspended quiescent cells; however, it decreases once replated and allowed to re-enter the cell cycle. This is seen by an increase in Cyclin D expression. Represented by one- way ANOVA with Tukey’s post hoc test. (C) Representative blots. Data are presented as mean SD of n = 3, * = P ≤0.05.

Article Snippet: The following primary antibodies were used: FKBP25 (WB; 1 : 3000, MAB3955, R&D Systems, Minneapolis, MN, USA), FKBP25 (IF; 1 : 500, ab16654, Abcam, Waltham, MA, USA), fast myosin heavy chain (1 : 3000, Ab51263, Abcam), myogenin (1 : 3000, Ab134175, Abcam), detyrosinated alpha-tubulin (1 : 1000, ab131368, Abcam), MyoD1 (1 : 1000, #13812, CST, Danvers, MA, USA), Cyclin D1 (1 : 3000, #55506, CST), beta-actin (1 : 5000, #4970, CST), Stathmin (1 : 1000, #13655, CST), acetylated alpha-tubulin (Lys40; 1 : 3000, #32–2700, Invitrogen), total alpha-tubulin (1 : 5000, sc5286, Santa Cruz, Dallas, TX, USA) and pan tubulin (1 : 1000, #ATN02, Cytoskeleton, Denver, CO, USA).

Techniques: Suspension, Expressing

Journal: The FEBS journal

Article Title: FKBP25 regulates myoblast viability and migration and is differentially expressed in in vivo models of muscle adaptation.

doi: 10.1111/febs.16894

Figure Lengend Snippet: Fig. 5. FKBP25 knockdown does not impact upon features of myogenic differentiation or expression of myogenic regulatory factors. Proliferating C2C12 myoblasts were transduced with lentiviral particles containing a plasmid expressing a doxycycline (Dox)-inducible shRNA (Mir2) that targets FKBP25 mRNA to knockdown FKBP25 protein expression or a non-targeting (NT) shRNA as the control. Transduced cells were selected and passaged every 2–3 days, for 10 days. To induce FKBP25 knockdown in C2C12 myotubes, myoblasts were treated with 0.5 lgmL1 doxycycline (Dox) in growth medium for 72 h prior to transition to differentiation medium containing 2% horse serum for up to 96 h. (A) 25KD knockdown in proliferating myoblasts was maintained throughout subsequent C2C12 differentiation. (B–E) 25KD did not impact the protein expression of differentiation markers, fast myosin heavy chain, myogenin or MyoD in differentiated C2C12 myotubes. All data are presented as mean SD, n = 3, * = P ≤0.05.

Article Snippet: The following primary antibodies were used: FKBP25 (WB; 1 : 3000, MAB3955, R&D Systems, Minneapolis, MN, USA), FKBP25 (IF; 1 : 500, ab16654, Abcam, Waltham, MA, USA), fast myosin heavy chain (1 : 3000, Ab51263, Abcam), myogenin (1 : 3000, Ab134175, Abcam), detyrosinated alpha-tubulin (1 : 1000, ab131368, Abcam), MyoD1 (1 : 1000, #13812, CST, Danvers, MA, USA), Cyclin D1 (1 : 3000, #55506, CST), beta-actin (1 : 5000, #4970, CST), Stathmin (1 : 1000, #13655, CST), acetylated alpha-tubulin (Lys40; 1 : 3000, #32–2700, Invitrogen), total alpha-tubulin (1 : 5000, sc5286, Santa Cruz, Dallas, TX, USA) and pan tubulin (1 : 1000, #ATN02, Cytoskeleton, Denver, CO, USA).

Techniques: Knockdown, Expressing, Transduction, Plasmid Preparation, shRNA, Control

Journal: Molecular oncology

Article Title: SETD2 loss in renal epithelial cells drives epithelial-to-mesenchymal transition in a TGF-β-independent manner.

doi: 10.1002/1878-0261.13487

Figure Lengend Snippet: Fig. 7. SOX2, OCT2, and PRRX1 are downstream effectors of the SETD2-regulated EMT program. (A) Expression of SOX2, OCT2, and PRRX1 in TGF-b-treated WT (72 h), SETD2 KO, and SETD2 rescue tested by RT-qPCR (run in triplicate). (B) Migration capacity by wound healing assay, (C) invasiveness by transwell assay, and (D) stemness by 3D spheroid formation assay in RPTEC WT GFP (control vector), SETD2 KO1 and KO2, and SOX2/OCT2/PRRX1-transduced WT RPTEC lines. Images are taken at 49 magnification, scale bar: 1000 lm for (B) and (D) and at 2.59 magnification, scale bar: 1200 lm for (C). Data are represented as mean SEM for triplicate reactions for B–D. P-value is calculated by one-way ANOVA in (A), (B), and (D). Two-way ANOVA is used for statistical test for (C). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P ≥0.05. (E) Model of the SETD2 loss-driven EMT program through cell intrinsic (transcriptional) and cell extrinsic (paracrine) mechanisms.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense #ab1791), SOX2 (R&D Systems; #AF2018-SP), OCT2 (Thermo Fisher; #39-5400), PRRX1 (Novus Biologicals, St. Charles, MO, USA; #NBP1-06067), anti-FLAG (Sigma-Aldrich; #F1804), GAPDH (Cell Signaling Technology; #2118S), and lamin B1 (Proteintech; #12987-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Migration, Wound Healing Assay, Transwell Assay, Tube Formation Assay, Control, Plasmid Preparation