Fig 1.
Ubtor depletion promotes neurite outgrowth in hippocampal neurons and ld-PC12 cells.
(A) Neurite outgrowth in primary culture of rat hippocampal neurons. Dissociated hippocampal neurons were transfected with either Cy3-labeled negative control siRNA (NC) or Ubtor siRNA (Ubtor siKD) and then cultured in vitro for 36 hrs (HIV). Neurites were stained by the acetylated tubulin antibody. Transfected cells were indicated by the Cy3 fluorescence signals from the Cy3-labeled siRNAs. Scale bar, 50 μm. (B) Quantitative analysis of neurite outgrowth at 36 HIV. Neurite lengths were measured from 15 images for the NC, and 16 images for the Ubtor siKD groups, taken from 3 independent experiments. n = 241 and 288 for the NC and the Ubtor siKD group, respectively. t = 12.71, df = 527, P < 0.0001. (C) Immunoblot analysis of siRNA mediated knock down in hippocampal neurons. t = 8.427, df = 2, P = 0.0138. (D) NGF-induced neurite outgrowths in ld-PC12 cells transfected with either Cy3-labeled negative control siRNA (NC) or Ubtor siRNA (Ubtor siKD). Transfected cells were serum-starved overnight and treated with 50 ng/ml of NGF for 0 and 48 hours. Scale bar, 50 μm. (E) Quantitative analysis of neurite outgrowth at 48 hours post NGF treatment. Neurite outgrowth rates were calculated from 38 images for the NC, and 36 images for the Ubtor siKD groups, taken from four independent experiments. t = 9.827, df = 72, P < 0.0001. Neurite lengths of differentiated cells were measured in these images. n = 88 and 243 for the NC and the Ubtor siKD group, respectively. t = 7.721, df = 329, P < 0.0001. (F) qRT-PCR analysis of Ubtor expression levels. Expression levels relative to GAPDH levels are normalized to the untreated NC group. Three biological repeats, F(2, 6) = 181.1, P < 0.0001. Multiple comparison significance values are indicated on the graph. (G) Immunoblot analysis of siRNA mediated knock down in ld-PC12 cells. t = 14.94, df = 2, P < 0.01.
Fig 2.
Depletion of UBTOR promotes cell growth.
(A) Cell proliferation analyses of HEK293T and glioma U87MG cells transfected with negative control (NC) or UBTOR Lentiviral shRNAs (Lenti-1 or Lenti-2). Three biological repeats for each cell line. For the Lentiviral shRNA treatment factor, HEK293T: F(2, 6) = 116.1, P < 0.0001, and U87MG: F(2, 6) = 11.10, P < 0.0096. Multiple comparison significance values are indicated on the graph. (B) Immunoblot analysis of shRNA mediated knock down in HEK293T and U87MG cells. Three biological repeats for each cell line. For the Lentiviral shRNA treatment factor, HEK293T: F(2, 6) = 81.41, P < 0.0001, and U87MG: F(2, 6) = 180.4, P < 0.0001. Multiple comparison significance values are indicated on the graph. (C) Colony formation analysis of HEK293T cells with reduced UBTOR expression (Lenti-2) and control cells (NC). Transfected cells were plated at 500 cells/well in 6-well plate and grew for 7 days. Three biological repeats, t = 49.98, df = 4, P < 0.0001. (D) Colony formation analysis of T24 and HEK293T cells transfected with vector control (Vector) or UBTOR over-expressing construct UBTOR-FLAG-HA (UBTOROE). Transfected cells were plated at 5 x 104 (T24) or 2 x 104 (HEK293T) cells/plate in 6 cm plates and grew for 2 weeks. Three biological repeats, t = 12.73, df = 4, P < 0.001 for the T24 cells and t = 6.383, df = 4, P < 0.01 for the HEK293T cells.
Fig 3.
Ubtor depletion increases mTOR signaling.
(A) Immunoblot analysis of mTOR signaling pathway in ld-PC12 cells transfected with either negative control siRNA (NC) or Ubtor siRNA (Ubtor siKD). Transfected cells were serum starved overnight and treated with 50 ng/ml of NGF for 0 to 12 hours. In addition, cells were treated with 100 nM of rapamycin (rapa) or vehicle (DMSO) for 30 min after 12 hours of NGF treatment. GAPDH was used as a loading control. Representative results from four biological repeats. (B) Quantitative analysis of p-S6 levels. The p-S6/S6 values were normalized to that of the NC group. Four biological repeats. For the siRNA treatment factor, F(1, 6) = 88.21, P < 0.0001. Multiple comparison significance values across different time points are indicated on the graph. (C) Immunoblot analysis of mTOR signaling pathway in HEK293T cells transfected with Lentiviral shRNAs to reduce UBTOR expression. Transfected cells were serum starved overnight and then treated with 20% serum (FBS) with or without 100 nM of rapamycin for 30 min. The p-S6K/S6K values were normalized to that of the NC group. Three biological repeats. For the Lentiviral shRNA treatment factor, F(2, 6) = 13.66, P < 0.01. (D) ld-PC12 or HEK293T cells with reduced UBTOR expression were larger than control cells. Cell diameter distributions were measured in ld-PC12 or HEK293T cells transfected with negative control (NC) or siRNA (siKD) or shRNA (Lenti-1 or Lenti-2). Representative results from three biological repeats.
Fig 4.
Subcellular localization of UBTOR and mTOR complexes.
(A) Subcellular localization of UBTOR in HEK293T and hT-RPE cells, co-stained with endoplasmic reticulum (ER) marker PDIA4 or CANX. Figure inserts show enlarged views of the boxed area. Pearson correlation coefficient or Pearson’s r values are indicated on bottom left. Representative results from three biological repeats. Scale bar, 10 μm. (B) Subcellular localization of UBTOR and mTORC1 complex components in HeLa cells. Plasmid constructs encoding FLAG-tagged DEPTOR, myc-tagged RAPTOR, or FLAG-tagged MLST8 were co-transfected with EGFP-tagged UBTOR and immunostained with FLAG or myc antibodies respectively. Pearson’s r values are indicated on bottom left. Representative results from three biological repeats. Scale bar, 10 μm. (C) Serum or PAO treatment increases co-localization of UBTOR and endogenous mTOR protein. For serum treatment, transfected HeLa cells were first serum starved overnight and then treated with 20% FBS for 1 hour. For PAO treatment, transfected cells were treated with vehicle (DMSO) or 10 μM of PAO for 15 min. Quantitative analysis of co-localization is shown on the right. Representative results from 4 biological repeats. For the serum test, t = 3.776, df = 44, P = 0.005. For the PAO test, t = 3.742, df = 59, P = 0.0004. Scale bar, 10 μm.
Fig 5.
UBTOR interacts with mTOR complexes through DEPTOR.
(A) PAO treatment promotes interactions between UBTOR and endogenous mTOR complexes. HEK293T cells transfected with FLAG-tagged UBTOR were treated with 5 μM of PAO or vehicle (DMSO) for 15 min. Cell lysates were immunoprecipitated with anti-FLAG-M2 beads, and then probed with antibodies indicated on the figure panel. UBTOR interacted with DEPTOR with or without PAO treatment. Representative results from three biological repeats. Quantitative analysis of the immunoblots is shown on the right. Statistics significance values were indicated on the graph. (B) HA-tagged DEPTOR immunoprecipitates endogenous UBTOR. Quantitative analysis of the immunoblots is shown on the right. For DEPTOR-mTOR interaction test, t = 10.28, df = 2, P = 0.0093. For DEPTOR-UBTOR interaction test, t = 4.473, df = 2, P = 0.0465. (C) The N terminal region of UBTOR (UBTOR1-467) is sufficient to interact with endogenous DEPTOR and endogenous mTOR complexes. Experimental procedure similar to that of A. Representative results from three biological repeats. Quantitative analysis of the immunoblots is shown on the right. Statistics significance values were indicated on the graph. (D) The N terminal region of UBTOR (UBTOR1-467) directly interacts with the full length DEPTOR or the PDZ domain of DEPTOR. GST-tagged UBTOR1-467, his/HA double tagged DEPTOR, 2DEP domain, and PDZ domain proteins were expressed and purified from bacteria. Purified proteins were mixed, pulled-down by Nickle-beads, and immunoblotted as indicated. Representative results from three biological repeats. Quantitative analysis of the immunoblots is shown on the right. Statistics significance values are indicated on the graph.
Fig 6.
UBTOR stabilizes DEPTOR by regulating ubiquitination levels of DEPTOR.
(A) Immunoblot analysis of DEPTOR expression levels in ld-PC12 cells transfected with negative control (NC) and Ubtor siRNA. Transfected cells were treated with 50 ng/ml of NGF for 0 to 12 hours as indicated. Representative results from four biological repeats. Quantitative analysis of DEPTOR expression levels is shown on the right. The DEPTOR/GAPDH values were normalized to that of the NC group. Four biological repeats, Statistics significance values are indicated on the graph. (B) Immunoblot analysis of DEPTOR expression levels in HEK293T cells transfected with negative control (NC) and Ubtor Lentiviral shRNA (Lenti-1 and Lenti-2). Transfected cells were serum-starved overnight. Quantitative analysis was same as in A. Four biological repeats, F(2, 9) = 618.5, P < 0.0001. Multiple comparison significance values are indicated on the graph. (C) Immunoblot analysis of DEPTOR expression levels in HEK293T cells co-transfected with DEPTOR and various UBTOR constructs. UBTOR1-895: without transmembrane motif (Δ896–916). UBTOR1-851: without C terminal region (Δ852–916). Representative results from four biological repeats. Quantitative analysis of immunoblots is shown below. (D) The N terminal region (1–81) is required for UBTOR’s stabilization effect on DEPTOR. Representative results from three biological repeats. Quantitative analysis of immunoblots is shown below. (E) The middle region (468–773) of UBTOR is dispensable for UBTOR’s stabilization effect on DEPTOR. Representative results from three biological repeats. Quantitative analysis of immunoblots is shown below. (F) UBTOR overexpression reduces ubiquitination levels of DEPTOR in HEK293T cells. HA-tagged DEPTOR was co-transfected with either empty vector or FLAG-tagged UBTOR. Transfected cells were serum starved overnight, and then treated with 10% FBS and 20 μM MG132 for 6 hours. Cell lysates were immunoprecipitated with anti-HA beads, then probed with antibodies as indicated. Representative results from three biological repeats. Quantitative analysis of immunoblots is shown on the right. (G) Immunoblot analysis of DEPTOR ubiquitination levels. HEK293T cells were co-transfected as indicated. Representative results from three biological repeats. Quantitative analysis of immunoblots is shown on the right.
Fig 7.
Disruption of ubtor gene increases mTOR signaling in zebrafish brain tissues.
(A) Immunoblot analysis of p-S6K levels in wild type (wt) controls and ubtor-/- mutants. The head tissues were isolated from 5 dpf zebrafish before introducing food to fish larvae. For quantitative analysis, the p-S6K/S6K values were normalized to that of wt. t = 9.840, df = 2, P = 0.0102. Representative results from three biological repeats. (B) Immunoblot analysis of mTOR signaling in wt controls and ubtor-/- mutants under fasted and re-fed conditions. Zebrafish larvae of 13.5 dpf were starved for 36 hours, and then the larvae in the re-fed group were fed for 12 hours. Brain tissues were dissected out from the fasted and the re-fed larvae. For quantitative analysis, the p-S6K/S6K and p-S6/S6 values were normalized to that of wt. For p-S6K/S6K, t = 5.089, df = 2, P = 0.0385 under the fasted condition. For p-S6/S6, t = 4.637, df = 2, P = 0.0435 under the fasted condition, and t = 6.077, df = 2, P = 0.0260 under the re-fed condition. Representative results from three biological repeats. (C) Dominant-active HRAS(G12V) induced tumor formation in wild type controls (wt) and ubtor mutants. Three and four biological repeats for the wt and the mutant, respectively. t = 4.343, df = 5, P < 0.01. n = 59 and 37 for the wt and the mutant, respectively. χ12 = 13.23, P < 0.001.
Fig 8.
UBTOR depletion promotes xenograft tumor growth and mTOR signaling in nude mice.
(A) Xenograft tumor growth in nude mice. A total of 1 x 106 U87MG cells transfected with lentiviral shRNA (NC or UBTOR KD) were implanted into nude mice. At the endpoint, NC and KD tumors were removed and photographed. Results from two biological repeats (Trial-1 and Trial-2). (B) Quantitative analysis of the xenograft tumor volume growth. For the UBTOR-depletion KD factor, F (1, 13) = 9.79, P = 0.008. Multiple comparison significance values are indicated on the graph. (C) Representative H&E staining images of the xenograft tumors. Right panels (NC’ and KD’) show enlarged views of the boxed area on the left panels. Scale bar, 100 μm. (D) Ki-67 staining of the xenograft tumors. n = 4 for the NC and the KD xenograft tumors, respectively. A minimum of 2000 nuclear profiles were counted per tumor lesion. t = 13.35, df = 6, P < 0.0001. Scale bar, 100 μm. (E) Immunoblot analysis of UBTOR, S6 and p-S6 expression levels in xenograft tumors. S6 and p-S6 levels were normalized to that of β-actin, and then to that of NC. For S6, t = 14.9, df = 13, P < 0.0001. For p-S6, t = 6.613, df = 13, P < 0.0001.