Journal: Molecular and Cellular Biology

Article Title: Calcineurin Mediates the Gonadotropin-Releasing Hormone Effect on Expression of Both Subunits of the Follicle-Stimulating Hormone through Distinct Mechanisms

doi: 10.1128/mcb.06083-11

Figure Lengend Snippet: FIG. 3. The calcineurin target TORC1 also activates the GSU promoter and is required for both the GnRH and calcineurin effects. (A to C) T3-1 (A and C) or LT2 (B) cells were transfected with mGSU-luc (150 ng) (A and B) or hGSU-luc (100 ng) (C) together with hTORC1, -2, or -3 (50 ng) (A) or an hTORC1 expression vector (25 ng) (B and C) before cell harvest and luciferase assays, as in Fig. 1B. n 4 to 6. (A) The mean values were compared for all groups, and those means not significantly different (P 0.05) were assigned the same letter. (B and C) Pairwise comparison of means was carried out (*, P 0.05; ***, P 0.001). (D to F) T3-1 cells were transfected with hGSU-luc (D and F) or mGSU-luc (E) together with two siTORC1 sequences (250 ng each) or siGFP (500 ng). Cells were also transfected with CA-CnA (50 ng) (D) or exposed to GnRH (E and F), and luciferase assays were carried out as before. The effects of siTORC1 on overexpressed and endogenous TORC1 are shown by Western blotting, with Pol II as a loading control. (G and H) The effect of siTORC1 on endogenous mGSU expression is also shown by RT-PCR in T3-1 cells with or without GnRH (G) and by real-time quantitative PCR in GnRH-treated T3-1 and LT2 cells (H). Data were analyzed and are presented as for Fig. 1F. (I) Finally, mGSU-luc was transfected together with CREB or various CREB mutant expression vectors and/or mTORC1 (75 ng of each), and some of the cells were exposed to GnRH before luciferase assays were carried out, as above. n 4. ANOVA compared all means separately for untreated (capital letters) and treated (lowercase letters) cells, and results that are similar (P 0.05) share the same letter.

Article Snippet: Abs against CnA (AB1696; Chemicon International, Inc., Temecula, CA), NFAT2, -3, and -4 (sc-13033, sc-13036, and sc-8321, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), TORC1 and pTORC1 (C71D11 and 3359; Cell Signaling, Danvers, MA); FLAG (F3265; Sigma), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) and polymerase II (Pol II) (sc-20357 and sc-899, respectively; Santa Cruz Biotechnology), diluted 1:1,000 to 1:2,000 in TBST plus 1% BSA or nonfat milk, were used.

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Comparison, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: Calcineurin Mediates the Gonadotropin-Releasing Hormone Effect on Expression of Both Subunits of the Follicle-Stimulating Hormone through Distinct Mechanisms

doi: 10.1128/mcb.06083-11

Figure Lengend Snippet: FIG. 5. TORC1 constantly translocates between the cytoplasm and the nucleus and is N-terminally truncated prior to GnRH treatment. (A) GFP-tagged TORC1 was overexpressed in T3-1 cells, and 24 h later, some of the cells were treated with leptomycin B (LMB) for 30 min before image capture of the live cells. (B and C) Cells were treated for 2 h with GnRH and/or CsA (added 30 min before GnRH) before fractionation of cell extracts to cytoplasmic and nuclear fractions. Each fraction was analyzed by Western blot analysis (with 10 [B] or 15 [C] g of protein/lane) for TORC1, with GAPDH as a loading control. (D) LT2 cells were treated and fractionated similarly, and levels of phospho- TORC (pTORC) or total TORC were assessed by Western blotting. In the center and right blots, FLAG-TORC and TORC-myc were transfected, respectively, 48 h before the treatments. Only cytoplasmic fractions are shown, because pTORC was undetectable in nuclear fractions. (E) LT2 cells were exposed to GnRH for 0, 30, or 120 min before Western blotting of the cell extract (in RIPA buffer; 10 g protein/lane) with an Ab to TORC1. Pol II was used as a loading control. The relative levels of the upper to the lower band (arrows) for each TORC doublet ( standard errors of the means) are shown. Asterisks indicate significant differences (*, P 0.05; **, P 0.01) from the ratio in untreated samples. (F) The expression of the FLAG-TORC construct in T3-1 cells was analyzed following extraction in nuclear lysis buffer, using an Ab to FLAG, without GnRH (0 h) or after 4 h of GnRH treatment. (G) Cherry-TORC1 was overexpressed in T3-1 cells, and after 4 h of GnRH treatment, cell compartments were separated before loading of 10 g protein/lane and blotting with an anti-TORC1 Ab. The upper bands, representing Cherry-TORC, increased 1.5-fold (nuclear fraction) or 1.75-fold (cytoplasmic fraction) in intensity over the lower TORC bands following GnRH treatment. (H and I) T3-1 (H) or LT2 (I) cells were transfected with Cherry-TORC1, and after 24 h, cells were exposed to 10 M MG132, 26 M calpain inhibitor (CI), and/or 20 M caspase inhibitor (VAD) for 4 h, before blotting of cell extracts (RIPA buffer; 15 g of protein/lane) with anti-TORC1 and anti-Pol II Abs.

Article Snippet: Abs against CnA (AB1696; Chemicon International, Inc., Temecula, CA), NFAT2, -3, and -4 (sc-13033, sc-13036, and sc-8321, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), TORC1 and pTORC1 (C71D11 and 3359; Cell Signaling, Danvers, MA); FLAG (F3265; Sigma), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) and polymerase II (Pol II) (sc-20357 and sc-899, respectively; Santa Cruz Biotechnology), diluted 1:1,000 to 1:2,000 in TBST plus 1% BSA or nonfat milk, were used.

Techniques: Fractionation, Western Blot, Control, Transfection, Expressing, Construct, Extraction, Lysis

Journal: Molecular and Cellular Biology

Article Title: Calcineurin Mediates the Gonadotropin-Releasing Hormone Effect on Expression of Both Subunits of the Follicle-Stimulating Hormone through Distinct Mechanisms

doi: 10.1128/mcb.06083-11

Figure Lengend Snippet: FIG. 6. Calcineurin is required for the GnRH effect on the activation of the FSH gene. (A) LT2 cells were transfected with mFSH-luc, and in some cells CnA was overexpressed before treatment with GnRH and/or CsA, after which luciferase assays were performed. Data are presented as above, except that means are shown relative to the same treatment group (FSH, CsA, or CnA). n 4. Pairwise analysis compared means within the same treatment group for cells with or without GnRH (NS, P 0.05; **, P 0.01; ***, P 0.001). (B) LT2 cells were either left untreated or treated with GnRH for as long as 60 min, and ChIP was carried out using an Ab against Pol II, endogenous NFAT3 (left), or FLAG (after overexpression of FLAG-tagged NFAT) (right). The presence of the Nur77 promoter was assessed by PCR. Also shown is amplification from the input samples and rabbit IgG controls. (C) RT-PCR was carried out in LT2 cells following transfection of siRNA to NFAT3, NFAT4, or GFP (as a control), with or without GnRH treatment, before amplification of a 380-bp fragment of the Nur77 coding sequence. (D) Luciferase assays were carried out in LT2 cells after transfection of FSH-luc either with siNFAT3 or with siGFP (300 ng) as a control. Data are presented as before. n 4. (E) Quantitative real-time PCR was used to measure endogenous levels of FSH or Nur77 mRNA in LT2 cells after transfection of siNFAT3 (right) or siGFP as a control (left). Levels are expressed relative to those in siGFP-transfected, GnRH-treated cells. Means standard errors of the means (n 3) are shown. (F) LT2 cells were transfected with FSH-luc together with mTORC1 and/or Nur77, and some of the cells were treated with GnRH. Luciferase assays were carried out, and data are presented as above. n 4. (G) LT2 cells were transfected with FLAG-TORC1 and were either left untreated or exposed to GnRH for 4 h. IP was then carried out using an Ab against FLAG, and the precipitated proteins were analyzed by Western blot analysis using an Ab against TORC1 or Nur77.

Article Snippet: Abs against CnA (AB1696; Chemicon International, Inc., Temecula, CA), NFAT2, -3, and -4 (sc-13033, sc-13036, and sc-8321, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), TORC1 and pTORC1 (C71D11 and 3359; Cell Signaling, Danvers, MA); FLAG (F3265; Sigma), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) and polymerase II (Pol II) (sc-20357 and sc-899, respectively; Santa Cruz Biotechnology), diluted 1:1,000 to 1:2,000 in TBST plus 1% BSA or nonfat milk, were used.

Techniques: Activation Assay, Transfection, Luciferase, Over Expression, Reverse Transcription Polymerase Chain Reaction, Control, Sequencing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Calcineurin Mediates the Gonadotropin-Releasing Hormone Effect on Expression of Both Subunits of the Follicle-Stimulating Hormone through Distinct Mechanisms

doi: 10.1128/mcb.06083-11

Figure Lengend Snippet: FIG. 7. TORC1 increases the GnRH activation of the FSH promoter and works synergistically with Nur77 and/or CREB. (A) LT2 cells were transfected with FSH-luc; some were also transfected with CREB, CA-CREB, or CREB S133A; and some were treated with GnRH, after which luciferase assays were performed as above. Means of absolute firefly luciferase levels are shown (n 4). (B) FSH-luc was transfected with TORC1 either alone or together with CREB, CA-CREB, or CREB S133A, and some of the cells were exposed to GnRH. Levels of firefly luciferase were normalized to those of Renilla luciferase, and the results are expressed as fold induction over the activity in control untreated cells. n 4. (C and D) LT2 cells were transfected with FSH-luc together with TORC1, Nur77, and/or WT-CREB (C) or with FSH-luc together with TORC1, Nur77, and/or WT-CREB, CREB S133A, or CREB S133E (D). n 4. For panels A to C, ANOVA compared all means separately for untreated (capital letters) and treated (lowercase letters) cells, and those that are similar (P 0.05) share the same letter. In panel D, pairwise analysis was carried out as indicated (NS, not significant; **, P 0.01). (E) ChIP was carried out in LT2 cells using an Ab against myc following transfection of TORC1-myc with or without Nur77, WT-CREB, CREB S133A, or CREB S133E, and the levels of the precipitated FSH promoter were assessed by real-time quantitative PCR. Results for two repeats are shown. Cytomegalovirus (CMV) was used as a control. (F) LT2 cells were transfected with a DN-TORC expression vector and FSH-luc, and some of the cells were treated with GnRH. Luciferase assays were carried out, and normalized luciferase levels relative to the levels in untreated control cells are presented. n 4. (G, H, and I) Alternatively, cells were transfected with siTORC, and some were treated with GnRH. (G) The effects on transfected FSH-luc levels were assessed by a luciferase assay. (H and I) The effects on endogenous FSH mRNA levels were measured by RT-PCR (H) or real-time quantitative PCR (I). In panel I, mRNA levels are expressed relative to those in untreated cells for each group (siGFP or siTORC). Statistical analysis was carried out as described above. n 2 to 4.

Article Snippet: Abs against CnA (AB1696; Chemicon International, Inc., Temecula, CA), NFAT2, -3, and -4 (sc-13033, sc-13036, and sc-8321, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), TORC1 and pTORC1 (C71D11 and 3359; Cell Signaling, Danvers, MA); FLAG (F3265; Sigma), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) and polymerase II (Pol II) (sc-20357 and sc-899, respectively; Santa Cruz Biotechnology), diluted 1:1,000 to 1:2,000 in TBST plus 1% BSA or nonfat milk, were used.

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Control, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: Inhibition of TORC1 reduces mating GPCR levels at the PM and represses the mating pathway. A - D , representative images of cells expressing ( A ) Ste2-mEnvy, ( B ) Ste3-mEnvy, ( C ) Gpr1-mEnvy, and ( D ) Pma1-mRuby2 treated with or without 0.2 μM rapamycin for 2 h. Scale bars represent 4 μm. E , quantification of Ste2-mEnvy, Ste3-mEnvy, Gpr1-mEnvy, and Pma1-mRuby2 mean fluorescent intensities at the plasma membrane (-Rap: Ste2 n = 212 cells, Ste3 n = 285 cells, Gpr1 n = 197 cells, Pma1 n = 270 cells. +Rap: Ste2 n = 277 cells, Ste3 n = , Gpr1 n = 214 cells, Pma1 n = ). Measurements were normalized to the mean of the untreated groups to produce percentages. Error bars represent ±SEM. ∗ = p < 0.05, ∗∗ = p < 0.01, and ∗∗∗ = p < 0.001. F , β-galactosidase (pFUS1-LacZ) mating transcription assays of cell cultures pretreated with or without 0.2 μM rapamycin for 2 h (n > 10 colonies). Error bars represent ±SEM. EC 50 and E max are reported with ±95% confidence intervals. G , quantitative mating assays in cells pretreated with or without 0.2 μM rapamycin for 2 h (n = 5 assays). Error bars represent ±SEM. ∗ = p < 0.05 by two-sample one-tailed unequal variance t test. GPCR, G-protein–coupled receptor; mEnvy, monomeric Envy; PM, plasma membrane; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Inhibition, Expressing, Clinical Proteomics, Membrane, One-tailed Test

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: TORC1 inhibition leads to CME of Ste2 through α-arrestins and Ypk1. A , to identify candidates involved in TORC1-mediated endocytosis, we deleted multiple genes that are involved in TORC signaling, receptor endocytosis, and CME. Casein kinases ( , , ), α-arrestins ( , , , ), and epsin-like proteins ( , ) are all involved in priming the pheromone receptors for CME during mating. CME utilizes motors and cytoskeletal organizers ( , ) to form then endocytic pit that matures into an endosome. During starvation, TORC1 activity is reduced, downregulating PP2A activity . TORC2 activates Ypk1 and Ypk2 ( , , ), which repress major cell regulators such as calcineurin . B , quantification of mean Ste2-mEnvy on the plasma membrane in the indicated strain, with or without 0.2 μM rapamycin for 2 h (n > 170 cells). Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA was used to analyze variance between treated and untreated mutant and WT cells. Tukey’s HSD test was used as a multiple comparison test to compare means between groups. Based on ANOVA results, genetic mutants treated with rapamycin were grouped into one of three categories: no rescue ( red ), partial rescue ( yellow ), and complete rescue ( green ). Mutants in the no rescue category were only significantly different ( p < 0.05) from untreated WT cells. Mutants in the partial rescue category were significantly different from both treated and untreated WT cells. Mutants in the complete rescue category were only significantly different from rapamycin-treated WT cells. Error bars represent ±SEM. Bars with dashed outlines show data reported in E . C – G , representative images of ( C ) WT, ( D ) end 3Δ, ( E ) rog Δ, ( F ) rod 1Δ, and ( G ) ypk 1Δ cells expressing Ste2-mEnvy treated with or without 0.2 μM rapamycin for 2 h. Scale bars represent 4 μm. CME, clathrin-mediated endocytosis; HSD, honestly significant difference; mEnvy, monomeric Envy; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Inhibition, Activity Assay, Clinical Proteomics, Membrane, Mutagenesis, Comparison, Expressing

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: The C terminus of Ste2 is required for TORC1-mediated endocytosis. A , representative images of cells expressing Ste2-mEnvy and Ste2 T326 -EGFP treated with (n = 184 cells) and without (n = 132 cells) 0.2 μM rapamycin for 2 h. Scale bar represents 4 μm. B , quantification of Ste2-mEnvy and Ste2 T326 -EGFP mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. Error bars represent ±SEM. ∗ = p < 0.001. Bars with dashed outlines show data reported in E . HSD, honestly significant difference; mEnvy, monomeric Envy; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: The Ypk1 activators Pkh1 and Pkh2 contribute to TORC1-mediated Ste2 endocytosis. A , representative images of pkh1Δ cells expressing Ste2-mEnvy treated with (n = 186 cells) or without (n = 195 cells) 0.2 μM rapamycin for 2 h. B , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. C , representative images of pkh2Δ cells expressing Ste2-mEnvy treated with (n = 135 cells) or without (n = 162 cells) 0.2 μM rapamycin for 2 h. D , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. All error bars represent ±SEM. ∗ = p < 0.01, ∗∗ = p < 0.001. All bars with dashed outlines show data reported in E . Scale bars represent 4 μm. mEnvy, monomeric Envy; HSD, honestly significant difference.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: TORC2 activity directs TORC1-mediated Ste2 endocytosis. A , representative images of cells expressing Ste2-mEnvy and avo3 T1273 treated with (n = 233 cells) and without (n = 139 cells) 0.2 μM rapamycin for 2 h. B , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. C , representative images of cells expressing Ste2-mEnvy and avo3 T1273 grown in SCD media (+N) (n = 141 cells) or low nitrogen SCD media (-N) (n = 109 cells) for 6 h. D , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. All error bars represent ±SEM. ∗ = p < 0.05, ∗∗ = p < 0.001. All bars with dashed outlines show data reported in E . Scale bars represent 4 μm. HSD, honestly significant difference; mEnvy, monomeric Envy; SCD, Synthetic Complete plus Dextrose; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Activity Assay, Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: Proposed mechanism of TORC1-directed endocytosis. In nutrient-deprived conditions or during rapamycin-mediated inhibition, TORC1 is repressed, leading, through an unknown pathway, to the TORC2-directed activation of Ypk1. Active Ypk1 facilitates α-arrestin–directed CME of Ste2, which ultimately traffics to the vacuole. During the pheromone response, TORC2 is also required for endocytosis, after which Atg8 promotes trafficking of the receptor to the lumen of the vacuole. CME, clathrin-mediated endocytosis; mEnvy, monomeric Envy; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Inhibition, Activation Assay

Journal: Open Biology

Article Title: Human gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signalling in budding yeast

doi: 10.1098/rsob.220366

Figure Lengend Snippet: The NTDs of GSDMD and MLKL cause a cell cycle arrest through the inhibition of TORC1. ( a ) Cell cycle profiles obtained by measuring DNA content (FL2-A) of cells stained with PI and subsequently analysed by flow cytometry ( n = 10 000) (left panels), and graph showing the percentage of cells in phase G0/G1 for each population (right panel) of BY4741 strain bearing plasmids as in b , f , after 5 h of induction in SG medium. ( b ) Immunoblot showing Sch9 phosphorylation (upper panel) and quantification of P-Sch9 relative to total Sch9 (lower panel) in yeast lysates of BY4741 strain bearing the plasmids pJU676 (Sch9-5xHA) and pAG413-GSDMD-EGFP, pAG413-GSDMD(NT)-EGFP, pAG413-MLKL-EGFP, pAG413-MLKL(PM)-EGFP or pAG413-MLKL(1–182)-EGFP after 7 h of induction in SG medium. pAG413-EGFP empty vector was used as a control. ( c ) Immunoblot showing Atg13 phosphorylation (upper panel) and quantification of P-Atg13 relative to total Atg13 (lower panel) in yeast lysates of BY4741 strain bearing the plasmids HC078 (3xHA-Atg13) and the same plasmids as in ( b ). ( d ) Immunoblot showing Maf1 phosphorylation (upper panel) and quantification of P-Maf1 relative to total Maf1 (lower panel) in yeast lysates of BY4741 strain bearing plasmids pAH099 (Maf1-3xHA) and the same plasmids as in ( b ). In ( b–d ), cells treated with 100 nM rapamycin for 5 h were used as a positive control of TORC1 inhibition. Membranes were hybridized with anti-HA antibody. Anti-G6PDH antibody was used for loading control. A representative blot from three different experiments with different transformants is shown. In ( a–d ), results correspond to the mean of three biological replicates performed on different transformants. Error bars represent the standard deviation. Asterisks (*, **, ***) indicate p -values of < 0.05, < 0.01 and < 0.001, respectively, by Tukey's HSD test.

Article Snippet: HC078 (pRS315-3xHA-Atg13) plasmid, used also as a readout of TORC1 activity, was obtained from Addgene (Addgene_ 59544).

Techniques: Inhibition, Staining, Flow Cytometry, Western Blot, Phospho-proteomics, Plasmid Preparation, Control, Positive Control, Standard Deviation