TSH increased SREBP-2 protein levels and the expression of its target genes, HMGCR and HMGCS, in HepG2 cells.

<p>(A) HepG2 cells were pretreated with TSH (4 μM) for 24 or 48 h. Whole cell lysates were subjected to Western blotting (WB) using an SREBP-2 antibody that recognizes both the SREBP-2 precursor and nuclear active forms. (P) and (N) denote the precursor and nuclear active forms of SREBP-2, respectively. (B-C) Densitometric quantifications of SREBP-2 (P) and SREBP-2 (N) are shown. Densitometry was performed using ImageJ (version 1.45) and normalized to β-actin. The data are presented as the mean ± SEM. *<i>p</i>< 0.05 versus zero concentration of TSH, ^#<i>p</i> < 0.05 versus TSH (24h). (D) HepG2 cells were treated with TSH (4 μM) for 24 or 48 h and then were harvested to monitor the mRNA expression of HMGCR and HMGCS. β-actin was used for normalization, and the control was set to 1 in the Real-Time PCR data. All the experiments were performed in duplicate. *<i>p</i> < 0.05 versus zero concentration of TSH.</p

Quantitative RT-PCR primers.

<p>Quantitative RT-PCR primers.</p

AMPK activation by AICAR attenuated the TSH-mediated increase in SREBP-2.

<p>(A) TSH decreased AMPK activity. HepG2 cells were synchronized overnight in serum-free EMEM and then treated with AICAR (0.5 mM) or TSH (4 μmol/L) for 24 h. AMPK activity was measured using the SAMS peptide phosphorylation assay and was calculated as picomoles per minute per milligram protein. The data are presented as the mean ± SEM (n = 4). *<i>p</i> < 0.05 compared with control (con). ^#<i>p</i> < 0.05 versus TSH. (B) HepG2 cells were pretreated with TSH (4 μM) for 24 h in the absence or presence of AICAR (0.5 mM) for 24 h. Whole cell lysates were subjected to WB using the SREBP-2 antibody. (C-D) Densitometric quantification of SREBP-2 (P) and SREBP-2 (N). The data are presented as the mean ± SEM. *<i>p</i> < 0.05 versus untreated cells; ^#<i>p</i> < 0.05 versus TSH-treated cells. (E) AICAR activated AMPK, which down-regulated the mRNA expression of HMGCR and HMGCS in TSH-treated HepG2 cells. These experiments were repeated at least three times with similar results.</p

AMPK activation by AICAR induced the threonine phosphorylation of the precursor and nuclear forms of SREBP-2.

<p>(A) HepG2 cells were transfected with pc-DNA3.1 encoding 2×flag-tagged human nuclear SREBP-2 or pcDNA empty vector or vehicle for 48 h and then were treated with or without AICAR (1 mM) for 24 h. Cell lysates were purified by immunoprecipitation (IP) with an anti-Flag antibody and were subjected to WB with antibodies against phosphorylated-Threonine (p-Thr) or phosphorylated-Serine (p-Ser). Total lysates were analyzed by WB with anti-flag and SREBP-2 antibodies as indicated. (B) HepG2 cells were treated with AICAR (1 mM) for 24 h, and the cell lysates were immunoprecipitated with an SREBP-2 antibody and subjected to WB with antibodies against p-Thr and p-Ser (data not shown). SREBP-2(P) denotes the antibody that only recognizes the SREBP-2 precursor. (C) Total protein (500 μg) from <i>Tshr</i>^<i>+/+</i> and <i>Tshr</i>^<i>-/-</i> mouse liver extracts were purified by IP with the SREBP-2 precursor antibody and subjected to WB with the p-Thr antibody. Total lysates were analyzed by WB with antibodies against p-AMPK and SREBP-2 as indicated. (D) Lysates from L02 cells treated with AICAR for 24 h were subjected to IP with an SREBP-2 antibody and then analyzed by WB with an antibody against p-Thr.</p

AICAR-activated AMPK decreased SREBP-2 protein levels.

<p>(A) HepG2 cells were treated with or without 0.5 mmol/l (0.5 mM) AICAR for 12 or 24 h and were subsequently washed with phosphate-buffered saline (PBS) and lysed. Nuclear and cytoplasmic extracts were analyzed by WB. β-actin was used as a cytoplasmic marker, and LaminB1 (LMNB1) was used as the nuclear marker. (B-C) Densitometric quantification of SREBP-2 (P) and SREBP-2 (N). The data are presented as the mean ± SEM. *<i>p</i>< 0.05 versus control (con, without AICAR). (D) AICAR-activated AMPK inhibited the expression of SREBP-2 in HepG2 cells. HepG2 cells were treated with or without 0.5 mM AICAR for 12 or 24 h and then were harvested to determine the mRNA expression of SREBP-2. β-actin was used for normalization, and the control was set to 1 in the Real-Time PCR data. All the experiments were performed in duplicate. *<i>p</i> < 0.05 versus control. (E) Immunofluoresence images of SREBP-2 (red) and nuclear staining with 4', 6-diamidino-2-phenylindole (DAPI, blue) in HepG2 cells. Magnification, ×200. Semiquantification analysis by ImageJ software of fluorescence intensity of SREBP-2 precusor and nuclear form in HepG2 cells. Bar-graph represents the results from 3 separate experiments and the fluorescence intensity of SREBP-2 from HepG2 cells without AICAR treatment was set as 1.</p

Double immunofluorescence staining for phosphorylated SREBP-2 in HepG2 cells.

<p>HepG2 cells were treated with AICAR (1 mmol/L) for 12 h or TSH (4 μM) for 24 h. The yellow in the images is derived from dual staining with the red p-Thr antibody and the green anti-SREBP-2 antibody. The nuclei were detected with 4', 6-diamidino-2-phenylindole (DAPI). Original magnification, The intensity of staining obtained with SREBP-2/p-Thr was measured by ImageJ.</p

Proposed regulatory mechanism of SREBP-2 by TSH and AMPK.

<p>Proposed regulatory mechanism of SREBP-2 by TSH and AMPK.</p

Additional file 2: of Long non-coding RNAs of switchgrass (Panicum virgatum L.) in multiple dehydration stresses

Table S1. List of memory lncRNAs. Table S2. Primers used in qRT-PCR. Table S3. Expression summary of different samples and treatments. Table S4. List of differentially expressed lncRNAs. Table S5. LncRNAs annotated by antisense and upstream or downstream. Table S6. Differentially expressed lncRNAs and the annotated genes. Table S7. Differentially expressed lncRNAs annotated with differentially expressed genes. Table S8. GO term _ response to stress. Table S9. Top 20 pathway enriched in annotation genes. Table S10. LncRNAs and genes involved in ABA biosynthesis and signal transduction. Table S11. LncRNAs and genes involved in ETH biosynthesis and signal transduction. Table S12. LncRNAs and genes involved in starch and sucrose metabolism. Table S13. Dehydration response and transcriptional memory genes or lncRNAs in switchgrass, maize and Arabidopsis. (ZIP 3778 kb

MOESM3 of Transcriptional and physiological data reveal the dehydration memory behavior in switchgrass (Panicum virgatum L.)

Additional file 3: Table: S2. Primers used in qRT-PCR

MOESM6 of Transcriptional and physiological data reveal the dehydration memory behavior in switchgrass (Panicum virgatum L.)

Additional file 6: Table S5. Differentially expressed genes involved in JA biosynthesis and signal transduction