Journal: The EMBO Journal

Article Title: LncRNA LITATS1 suppresses TGF‐β‐induced EMT and cancer cell plasticity by potentiating TβRI degradation

doi: 10.15252/embj.2022112806

Figure Lengend Snippet: Interactions between LITATS1 and TGF‐β/SMAD signaling components or modulators were analyzed by RIP. RT–qPCR was performed to detect LITATS1 expression in immunoprecipitates from HEK293T cells transfected with expression constructs for the indicated proteins. Representative results from two independent experiments are shown. Interactions between LITATS1 and TβRI or SMURF2 in MDA‐MB‐231 cells were detected by the CARPID approach. Cells with stable expression of TurboID–dCasRx were transduced without (Co.) or with (#1 and #2) LITATS1 targeting gRNAs. Cells were stimulated with or without TGF‐β (2.5 ng/ml) for 2 h and were then stimulated with biotin (500 μM) for 30 min. Western blotting was performed to detect SMURF2 and TβRI expression in whole‐cell lysates (Input) and immunoprecipitates (IP). GAPDH and HA‐dCasRx expression levels were measured for equal loading of Input samples and as the negative control or positive control, respectively, for proximity biotinylation in immunoprecipitate (IP) samples. Representative results from a minimum of three independent experiments are shown. Interactions between LITATS1 and TβRI (left) or SMURF2 (right) were analyzed by RIP. MDA‐MB‐231 cells were stimulated with or without TGF‐β (5 ng/ml) for 4 h before RIP. RT–qPCR was performed to detect LITATS1 expression in immunoprecipitates from MDA‐MB‐231 cells. IgG was included as the control for immunoprecipitation. Representative results from two independent experiments are shown. Interactions between LITATS1 and caTβRI or SMURF2 were analyzed by RNA pull‐down. Biotinylated 25 x poly(A), antisense LITATS1 ( LITATS1‐AS ), or LITATS1 was incubated with lysates from HEK293T cells transfected with the caTβRI‐HA or MYC‐SMURF2 expression construct. Western blot analysis was performed to detect HA or MYC expression in whole‐cell lysates (Input) and immunoprecipitates (IP). Representative blots from a minimum of three independent experiments are shown. In vitro RNA pull‐down assays were performed to evaluate the interactions between LITATS1 and SMURF1/2. In vitro ‐transcribed antisense LITATS1 ( LITATS1 ‐ AS ) or LITATS1 ( LITATS1 ‐ S ) was incubated with recombinant FLAG‐tagged SMURF1 or SMURF2 protein. Western blotting analysis was performed to evaluate FLAG expression in input and IP samples. The amounts of RNA probes used for RNA pull‐down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown. Quantification of TβRI‐SMURF2 PLA in A549 cells with or without LITATS1 knockdown were treated with or without TGF‐β (5 ng/ml) for 2 h. Representative images are shown in Fig . Effect of LITATS1 overexpression on SMURF2‐mediated TβRI polyubiquitination. HEK293T cells were transfected with expression constructs for HA‐Ubiquitin (HA‐Ub) and caTβRI‐FLAG and ectopic expression constructs for SMURF2 and/or LITATS1 . Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown. Effect of SMURF2 knockdown on LITATS1 ‐mediated TβRI polyubiquitination. MDA‐MB‐231 cells with stable HA‐Ub expression were transduced with expression constructs for LITATS1 and/or two different SMURF2 shRNAs, as indicated. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown. Data information: (C) is expressed as the mean ± SD values from three ( n = 3) biological replicates. (F) is expressed as the mean values from 15 ( n = 15) biological replicates. **0.001 < P < 0.01; ***0.0001 < P < 0.001; **** P < 0.0001. Statistical analysis was based on the unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: To identify interactions between lncRNAs and proteins of interest, RIP was performed with a Magna RIPTM RNA‐Binding Protein Immunoprecipitation Kit (Merck Millipore; 17‐700).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Construct, Western Blot, Negative Control, Positive Control, Control, Immunoprecipitation, Incubation, In Vitro, Recombinant, Agarose Gel Electrophoresis, Knockdown, Over Expression, Ubiquitin Proteomics, Transduction