Article Figures & Data
Figures
- Figure 1. TRINGS is upregulated by p53
Total RNA from U2OS cells treated with or without doxorubicin was subjected to Northern blot analysis to determine the molecular size of lncRNA TRINGS. 28S and 18S RNA were used as loading controls.
H1299 cells with doxycycline‐inducible expression of wild‐type p53 (H1299 Tet‐On p53) were incubated with doxycycline (1 μg/ml) for the indicated periods of time. Cell lysates were analyzed by Western blotting with indicated antibodies; total RNA was analyzed by real‐time RT–PCR analysis for lncRNA TRINGS and lncRNA DINO. Data shown are mean ± SD (n = 3).
Levels of TRINGS from different cell lines including HAFF, HepG2, Mel‐CV, U87, SMCC‐7721, HCT116, and U2OS were compared by real‐time RT–PCR. Cell lysates were analyzed by Western blotting with indicated antibodies. Data shown are mean ± SD (n = 3).
RNA FISH was performed with Alexa Fluor® 488‐labeled probe (red) recognizing TRINGS in U2OS cells. The nucleus was stained by Hoechst (blue). The representative fluorescence images taken by confocal microscopy are shown.
HCT116 cells were incubated with doxorubicin to induce expression of p53. After 24 h, cell lysates and total RNA were subjected to Western blot and real‐time RT–PCR analyses, respectively. Data shown are mean ± SD (n = 3). HCT116 cells were infected with lentiviruses expressing control shRNA or p53 shRNA. 48 h after infection, total RNA and cell lysates were analyzed by real‐time RT–PCR and Western blot, respectively. Data shown are mean ± SD (n = 3).
U2OS cells were infected with lentiviruses encoding a shRNA against p53 or an irrelevant control shRNA. Cells were then treated with or without doxorubicin for 24 h. Total RNA and cell lysates were analyzed by real‐time RT–PCR and Western blotting, respectively. Data shown are mean ± SD (n = 3).
The copy number of TRINGS in U2OS cells treated with doxorubicin at the indicated time was determined. Data shown are mean ± SD (n = 3).
- Figure 2. TRINGS is a direct target of p53
U2OS cells were treated with PFT‐α for the indicated periods of time, and cell lysates were then analyzed by Western blotting with anti‐p53, anti‐p21, and anti‐Actin antibodies. Total RNA was analyzed by real‐time RT–PCR analyses. Data shown are mean ± SD (n = 3; ***P < 0.001).
H1299 cells were transfected with the plasmids encoding wild‐type p53 or indicated mutant forms of p53. 24 h after transfection, total RNA were then analyzed by real‐time RT–PCR analyses. The data are represented as mean ± SD (n = 3). Cell lysates were analyzed by Western blotting with anti‐p53, anti‐p21, and anti‐Actin antibodies (**P < 0.01).
Schematic illustration of putative p53 binding site in TRINGS gene promoter. pGL3‐based wild‐type and mutant reporter constructs used for luciferase assays are also shown. “mut” indicates the promoter region (−1,573 to −1,587 bp) with deletion of the functional p53 binding site.
U2OS cells lysates were analyzed by ChIP assay with anti‐p53 antibody or an isotype‐matched IgG. ChIP products were amplified by PCR with the indicated pairs of primers (Appendix Table S1).
U2OS cells were harvested, and cell lysates were subjected to ChIP analysis. The promoter fragments for p21 and TRINGS (−1,800 to −1,350 bp in the promoter) were amplified by quantitative real‐time PCR. Data are shown as the mean ± SD (n = 3).
U2OS cells were co‐transfected with either Flag‐p53 or control vector plus the TRINGS promoter (−1,800 to −1,350 bp) reporter constructs and Renilla luciferase plasmid. 24 h after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity (mean ± SD; n = 3; *P < 0.05).
U2OS cells were infected with dCas9‐KRAB and indicated guide RNA lentiviruses. After induction with doxycycline (1 μg/ml) for 48 h, total RNA was analyzed by real‐time RT–PCR analysis for TRINGS expression. Data shown are mean ± SD (n = 3; ***P < 0.001).
- Figure 3. TRINGS promotes cell survival under glucose starvation condition
A, B U2OS (A) or HAFF (B) cells were infected with control shRNA or p53 shRNA, and 48 h after infection, cells were cultured in 2.5 mM glucose medium for indicated time. Levels of TRINGS of U2OS (A) or HAFF (B) were analyzed using real‐time RT–PCR assay. Data shown are mean ± SD (n = 3) and ** indicates P < 0.01. Cell lysates were also analyzed by Western blotting with anti‐p53, anti‐p21, and anti‐Actin antibodies.
C. Fluorescence microscopy images of U2OS and HAFF cells cultured in 2.5 mM glucose starvation medium for 48 h and followed by double staining with PI and Hoechst 33342.
D, E HAFF (D) or U2OS (E) cells treated with lentivirus encoding control shRNA or p53 shRNA (shp53) were cultured in 2.5 mM glucose medium for 72 and 48 h respectively. Cells were then stained with PI and Hoechst 33342. The extent of cell death was expressed as the percentage of PI‐positive cells to Hoechst 33342‐positive cells. Bars represent means ± SD of three independent experiments, and *** indicates P < 0.001. Cell lysates were analyzed by Western blotting with indicated antibodies.
F. The knockdown efficiency of TRINGS in the cytosol or nucleus was detected by real‐time RT–PCR. Data shown are mean ± SD (n = 3).
G–I Lentiviral infected U2OS cells containing control shRNA (shctrl), shTRINGS‐1, or shTRINGS‐2 were cultured in 2.5 mM glucose medium for 36 h. Cells were then stained with PI and Hoechst 33342 and visualized by fluorescence microscope (H); PI and Hoechst 33342 double staining cells were considered as necrotic cells, and PI staining cells were counted under microscope from 300 Hoechst 33342 staining cells. The percentage of necrotic cells in both TRINGS and control knockdown cells was plotted. Bars represent means ± SD of three independent experiments, and *** indicates P < 0.001 (I); the efficiency of TRINGS knockdown was analyzed by real‐time RT–PCR assay. Data shown are mean ± SD (n = 3) (G).
J, K Lentiviral infected U2OS cells containing control shRNA (shctrl), shp53 or shp53 plus pSin‐TRINGS were cultured in 2.5 mM glucose medium for 48 h. Cells were then stained with PI and Hoechst 33342 and visualized by fluorescence microscope (J). PI staining cells were counted under microscope from 300 Hoechst 33342 staining cells. The percentage of PI‐positive cells in both TRINGS and control knockdown cells was then quantified. Bars represent means ± SD of three independent experiments, and ** indicates P < 0.01, *** indicates P < 0.001 (K).
- Figure 4. Glucose starvation causes necrotic cell death in U2OS
U2OS cells were treated with DMSO (control) or Doxorubicin (Doxo) for 16 h; U2OS cells expressing control shRNA, TRINGS shRNA‐1, shRNA‐2 were cultured in 25 mM glucose (mock) or 2.5 mM glucose medium (glucose starvation) for 32 h. Cell lysates were then analyzed by Western blot analysis using anti‐PARP, anti‐Caspase3, or anti‐Actin antibodies.
U2OS cells expressing control shRNA or TRINGS shRNA‐1 were cultured in 2.5 mM glucose medium with or without apoptosis inhibitor Z‐VAD (20 μM) for 32 h, and cells were then stained with PI and Hoechst 33342. PI staining cells were then counted from 300 Hoechst 33342 staining cells; bars represent means ± SD of three independent experiments.
Control and TRINGS knockdown U2OS cells were treated with DMSO (mock), Nec1, DFO, or CQ respectively, and cells were then stained with PI and Hoechst 33342. PI staining cells were counted in 300 Hoechst 33342 staining cells; bars represent means ± SD of three independent experiments.
Control and TRINGS knockdown U2OS cells were cultured in 2.5 mM glucose starvation medium for 32 h. Cells were then harvested, and the intracellular levels of ATP were determined by ATP Assay Kit (Beyotime) according to the manufacturer's protocol (***P < 0.001; error bars are ±SE, n = 3).
Control and TRINGS knockdown (shTRINGS‐1 and shTRINGS‐2) U2OS cells were incubated with 2.5 mM glucose starvation medium for 32 h. Both culture medium and cell lysates were then analyzed by Western blotting using anti‐HMGB1 antibody. HMGB1‐Ex indicates extracellular HMGB1 and HMGB1‐In indicates intracellular HMGB1.
Control and TRINGS knockdown (shTRINGS‐1 and shTRINGS‐2) U2OS cells were incubated with 2.5 mM glucose starvation medium for 32 h. Lactate dehydrogenase (LDH) release was then used to test natural cytotoxicity of treated cells by LDH Cytotoxicity Assay Kit (Promega) according to the manufacturer's protocol (***P < 0.001; error bars are ±SE, n = 3).
Necrosis of U2OS cells under glucose starvation conditions was observed under transmission electron microscope at the micrometer scale. The left are U2OS cells without glucose starvation.
Control and RIP1 knockdown (shRIP1‐1, shRIP1‐2, or shRIP1‐3) U2OS cells were incubated with 2.5 mM glucose starvation medium for 48 h. Cells were then stained with PI and Hoechst 33342. PI staining cells were counted in 300 Hoechst 33342 staining cells. Bars represent means ± SD of three independent experiments. Cell lysates were analyzed by Western blotting using anti‐RIP1 and anti‐Actin antibodies. n.s. indicates no significance.
- Figure 5. TRINGS interacts with STRAP
U2OS cells lysates were incubated with in vitro‐synthesized biotin‐labeled antisense and control sense DNA probes against TRINGS, and biotin pull‐down assay was performed. Precipitated complexes from biotin pull‐down assay were separated by SDS–PAGE and visualized by Coomassie brilliant blue staining. Gel‐separated proteins were analyzed by MALDI mass spectrometry. Putative RNA‐binding proteins were indicated.
U2OS cells lysates were incubated with in vitro‐synthesized biotin‐labeled antisense and control sense DNA probes against TRINGS, and biotin pull‐down assay was performed. The pull‐downed complexes were analyzed by Western blot analysis using anti‐p53, anti‐STRAP, anti‐CIP2A, and anti‐Actin antibodies; the same complexes were also used for semi‐quantitative PCR analysis to ensure the presence of TRINGS.
Lysates from U2OS cells expressing Flag or Flag‐STRAP were used for co‐immunoprecipitation (co‐IP) using anti‐Flag resin. Eluted products were used for examining TRINGS and STRAP by real‐time RT–PCR and Western blot analysis respectively (***P < 0.001; error bars are ±SE, n = 3).
Lysates from U2OS cells under normal condition or glucose starvation (2.5 mM, 32 h) were used for co‐immunoprecipitation (co‐IP) using lgG or anti‐STRAP coupled resin. Eluted products were used for examining TRINGS and STRAP by real‐time RT–PCR and Western blotting analysis respectively. The intensity of STRAP bands was quantified by densitometry analysis (*P < 0.05; **P < 0.01; ***P < 0.001; error bars are ±SE, n = 3).
Western blot analysis of STRAP in control and TRINGS knockdown U2OS cells. Actin was served as protein loading control. The intensity of STRAP bands was quantified by densitometry analysis.
Total RNA from control or TRINGS knockdown U2OS cells was analyzed by real‐time RT–PCR to determine the STRAP mRNA levels and validate the knockdown efficiency of TRINGS as well. Data shown are mean ± SD (n = 3).
U2OS cells were infected with dCas9‐KRAB and indicated guide RNA lentiviruses. After induction with doxycycline (1 μg/ml) for 48 h, lysates was analyzed by Western blotting.
Control or TRINGS knockdown U2OS cells were treated with 50 μg/ml of cycloheximide (CHX) for indicated times. Subsequently, cell lysates were analyzed by Western blotting with anti‐STRAP and anti‐Actin antibodies.
Control or TRINGS overexpression U2OS cells were treated with 50 μg/ml of cycloheximide (CHX) for indicated times. Subsequently, cell lysates were analyzed by Western blotting with anti‐STRAP and anti‐Actin antibodies.
U2OS cells were treated with 50 μg/ml of cycloheximide (CHX) for indicated times. Furthermore, cells were treated with vehicle or MG132 (10 μM) for 4 h before harvesting the samples for protein. Subsequently, cell lysates were analyzed by Western blotting with anti‐STRAP and anti‐Actin antibodies.
U2OS cells were transfected with Flag‐STRAP and HA‐Ub plasmids as indicated in the figure. Cells were treated with MG132 for hours before cell lysis. Ectopically expressed Flag‐STRAP was immunoprecipitated with anti‐Flag antibody followed by Western blotting with anti‐Flag and anti‐HA antibodies.
U2OS cells were transfected with Flag‐STRAP, HA‐Ub, and pSin‐TRINGS in combination as indicated. Cells were treated with MG132 for hours before cell lysis. Ectopically expressed Flag‐STRAP was immunoprecipitated with anti‐Flag antibody followed by Western blotting with anti‐Flag and anti‐HA antibodies.
Control or TRINGS knockdown U2OS cells were transfected with Flag‐STRAP, HA‐Ub in combination as indicated. Cells were treated with MG132 for hours before cell lysis. Ectopically expressed Flag‐STRAP was immunoprecipitated with anti‐Flag antibody followed by Western blotting with anti‐Flag and anti‐HA antibodies.
- Figure 6. TRINGS promotes cell survival through STRAP–GSK3β–NF‐κB signaling
U2OS cells infected with control shRNA (shctrl), TRINGS shRNA (shTRINGS), TRINGS shRNA plus STRAP shRNA (shTRINGS plus shSTRAP), or STRAP shRNA (shSTRAP) were incubated with 2.5 mM glucose starvation medium for 32 h. Cells were then stained with PI and Hoechst 33342 and visualized by fluorescence microscope. PI‐positive cells were counted from 300 Hoechst 33342‐positive cells. Bars represent means ± SD of three independent experiments, ** indicates P < 0.01. Cell lysates were analyzed by Western blotting using anti‐STRAP antibody.
Control or TRINGS knockdown U2OS cells lysates were used for co‐immunoprecipitation assay with anti‐STRAP coupled resin. The input and co‐immunoprecipitates products were analyzed by Western blotting using anti‐STRAP or anti‐GSK3β antibodies. The intensity of STRAP‐binding GSK3β bands was quantified by densitometry analysis.
Lysates of U2OS cells were incubated with in vitro‐synthesized biotin‐labeled sense or antisense DNA probes against TRINGS, and then, biotin pull‐down assay was performed. The precipitates were then analyzed by RT–PCR using primers against TRINGS and by Western blotting using anti‐STRAP, anti‐GSK3β, or anti‐RelA antibodies.
Control shRNA and STRAP shRNA knockdown U2OS cells were cultured in DMEM medium (high glucose) or 2.5 mM glucose starvation medium for 32 h. Cell lysates were then analyzed by Western blot analysis using anti‐STRAP, anti‐p‐Gsk3β (ser 9), anti‐GSK3β, and anti‐Actin antibodies. The p‐Gsk3β (ser 9) bands were quantified by densitometry analysis.
U2OS cells expressing control shRNA, TRINGS shRNA, or TRINGS shRNA plus STRAP shRNA were cultured in DMEM medium (high glucose) or 2.5 mM glucose starvation medium for 32 h. Cell lysates were then analyzed by Western blotting using anti‐p‐Gsk3β (ser 9), anti‐GSK3β, anti‐STRAP, or anti‐Actin antibodies. The p‐Gsk3β (ser 9) bands were quantified by densitometry analysis.
Control shRNA (shctrl) or TRINGS shRNA (shTRINGS) U2OS cells were incubated with 2.5 mM glucose starvation medium with or without 3 μM CHIR99021 (inhibitor of GSK3β activity) for 32 h. Cells were then stained with PI and Hoechst 33342 and visualized by fluorescence microscope. PI‐positive cells were counted in 300 Hoechst 33342‐positive cells and plotted. Bars represent means ± SD of three independent experiments (** indicates P < 0.01).
Control and TRINGS knockdown U2OS cells were incubated with 2.5 mM glucose starvation medium for 32 h. Cell lysates were then analyzed by Western blot analysis using anti‐NF‐κB‐p50, anti‐RelA, and anti‐Actin antibodies.
Cell lysates from control and TRINGS knockdown U2OS cells were analyzed by Western blot analysis using anti‐IKBα and anti‐Actin antibodies.
U2OS cells expressing shctrl, shTRINGS or shGSK3β, shTRINGS plus shGSK3β were co‐transfected with pGL‐NF‐κB reporter construct and Renilla luciferase plasmid, respectively. 12 h after transfection, the cells were incubated with 2.5 mM glucose starvation medium. Reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity (mean ± SD). Cell lysates were analyzed by Western blotting using anti‐GSK3β and anti‐Actin antibodies (* indicates P < 0.05, ** indicates P < 0.01).
- Figure 7. The mode of lncRNA TRINGS in protecting cancer cells from necrosis induced by glucose starvation
TRINGS is activated by p53 upon glucose starvation. Upregulated TRINGS competes with GSK3β for binding to STRAP, and thus diminishes the interaction between STARP and GSK3β, and eventually TRINGS activates NF‐κB to protect cancer cells against necrosis.
Supplementary Materials
Dataset EV1 [embj201696239-sup-0001-DatasetEV1.xlsx]
Table EV1 [embj201696239-sup-0003-TableEV1.xlsx]
