Long non-coding RNA HOTAIR regulates cytoskeleton remodeling and lipid storage capacity during adipogenesis

Figures

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  Figure 1. HOTAIR is upregulated during early adipogenesis. (a) Relative expression in G-ASCs and A-ASCs of PPARG2, normalized tomatched mature adipocytes, and of HOTAIR, normalized to proliferative G-ASCs (mean fold difference ± SD; *p < 0.05, **p < 0.005, ***p < 0.001, mixed effect analysis with Šídák's multiple comparisons test (G-ASC vs A-ASC) or Tukey's multiple comparisons test (time-course); ASCs n ≥ 3; adipocytes n ≥ 2). (b) Relative HOTAIR expression in proliferative ASCs vs. mature adipocytes from the same tissue (mean fold difference ± SD; ***p < 0.001, two-tailed paired t test; n = 5 adipose tissue depots from 3 independent donors). (c) RT-PCR analysis of HOTAIR expression in a control (SCR) and two stable HOTAIR KD G-ASC lines (shHOTAIR1 and 2) (mean fold difference ± SD; *p < 0.05, **p < 0.005, two-way ANOVA with Dunnett's multiple comparisons test; n ≥ 3). (d) Differential expression of the top 6000 highly expressed genes between SCR and two shHOTAIR G-ASC lines (adjusted p-values < 0.05, limma, n = 3). (e) Overrepresentation analysis of downregulated (red) and upregulated (blue) genes from (d) using GO, Wikipathways, Reactome and Hallmark gene sets from MSigDb (adjusted…

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  Figure 2. HOTAIR KD impairs refeeding-induced protein synthesis in proliferating G-ASCs. (a) SUnSET analysis of protein synthesis rates in basal, 24-h fasting (HBSS, 0.2% FFA-free BSA) and 24-h refeeding conditions in SCR, shHOTAIR1 and shHOTAIR2 G-ASCs (see Fig. S5 for original blots). (b) SUnSET quantification (*p < 0.05, ***p < 0.001, two-way ANOVA with Dunnett's multiple comparisons test;; n = 3). (c) RT-qPCR analysis of pre-rRNA (ETS1, 36S), 18S and 5S rRNA after 24-h refeeding (*p < 0.05, ***p < 0.001, two-way ANOVA with Dunnett's multiple comparisons test;; n = 3). (d) Western blot analysis of phospho-Thr389 P70S6K, total P70S6K, RAPTOR and γ-Tubulin protein levels after 24-h refeeding (see Fig. S6for original blots). (e) Quantification of phospho-Thr389 P70S6K/total P70S6K and RAPTOR/γ-Tubulin (*p < 0.05, two-tailed paired T test, n = 3). (f) RT-qPCR validation of transient HOTAIR KD in proliferating G-ASC (mean ± SD; *** p < 0.001, ANOVA with Holm-Sidak's multiple comparisons test; n = 7). (g) Immunostaining of Nucleolin in siSCR- and siHOTAIR1- and 2-transfected G-ASCs. Nuclei are stained with Hoechst. Scale bar: 10 µm. (h) Number of nucleoli per cell quantified from Nucleolin…

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  Figure 3. HOTAIR KD prevents cytoskeletal remodeling during adipogenesis.( a) Phalloidin staining of F-actin in D0 SCR and shHOTAIR G-ASCs. Nuclei are stained with Hoechst. Scale bar: 10 µm. (b) Quantification of coherence in F-actin fibers alignment in D0 SCR and shHOTAIR G-ASCs (***p < 0.0001, two-way ANOVA with Dunnett's multiple comparisons test; n = 3 experiments, with ≥ 4 fields measured per experiment, 5 measurements per field). (c) Phalloidin staining of F-actin (green) and immunostaining of PERILIPIN1 (magenta) in differentiated (D9) SCR and shHOTAIR G-ASCs. Nuclei are stained with Hoechst. Scale bar: 10 µm. (d) Relative expression of ACTA2 in differentiating scrambled control (SCR) and shHOTAIR G-ASCs, normalized to SCR D0 (mean fold difference ± SD; *p < 0.05, **p < 0.005, two-way ANOVA with Dunnett's multiple comparisons test; n ≥ 3). (e) Western blot analysis (see Fig. S7for original blots) and (f) quantification of αSMA protein levels in differentiating SCR and shHOTAIR G-ASCs (mean fold difference ± SD; *p < 0.05, two-way ANOVA with Tukey's multiple comparisons test; n = 3). γ-Tubulin is shown as a loading control.

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  Figure 4. HOTAIR KD impairs lipid storage in mature adipocytes.( a) Oil red O staining (20X) of differentiated (D9) SCR and shHOTAIR G-ASCs. LipidL droplet area analysis from PERILIPIN immunostainings of differentiated (D9) SCR and HOTAIR KD G-ASCs presented as (b) individual data scatter plot and mean (***p < 0.0001, two-way ANOVA with Dunnett's multiple comparisons test;; n = 3 experiments, with ≥ 3 fields measured per experiment) and (c) distribution frequency. (d) RT-qPCR analysis of adipogenic transcription factor gene expression at D9 (mean fold difference ± SD; *p < 0.05, **p < 0.005, ***p < 0.001, two-way ANOVA with Dunnett's multiple comparisons test; n = 4). (e) Western blot analysis of fatty acid synthase (FAS), PERILIPIN1, and FABP4 in differentiating SCR and shHOTAIR G-ASCs (representative blot from n = 3; see Fig. SS7for original blots). γ-Tubulin is shown as a loading control. (f) RT-qPCR analysis of glucose- and lipid uptake-related genes, de novo lipogenesis genes and mature adipocyte markers in differentiated (D9) SCR and shHOTAIR G-ASCs (mean fold difference ± SD; *p < 0.05, **p < 0.005, two-way ANOVA with Dunnett's multiple comparisons test; n = 4). (g) Fold induction…