Journal: Science advances

Article Title: Uncoupling of Akt and mTOR signaling drives resistance to Akt inhibition in PTEN loss prostate cancers.

doi: 10.1126/sciadv.adq3802

Figure Lengend Snippet: Fig. 1. While PTEN loss enhances sensitivity to combined Akt and AR Inhibition, specific molecular alterations drive resistance in PTEN-deficient models. (A) Cell viability on day 7 in PTEN wild-type (WT) and CRISPR PTEN–modified CWR22Pc-EP, PCa15, and PCa16 treated with either ipatasertib (500 nM) or DMSO was assessed. (B) Expression levels of pS6, pPRAS40, and PSA were analyzed by Western blot in PTEN WT and CRISPR PTEN–modified CWR22Pc-EP, PCa15, and PCa16 cells. Cells were treated with DMSO, enzalutamide (10 μM) overnight, or ipatasertib (500 nM) for 4 hours. (C) Cell viability on day 7 was evaluated in PCa1, PCa2, PCa3, PCa8, PCa11, PCa12, and LNCaP following treatment with enzalutamide (Enza; 10 μM), ipatasertib (Ipa; 500 nM), or DMSO. (D and E) The expression levels of pS6, pPRAS40, and PSA in LNCaP, PCa1, PCa3, PCa2, PCa12, PCa11, and PCa8 were evaluated via Western Blot analysis. The cells were treated with DMSO, enzalutamide (10 μM) overnight, ipatasertib (500 nM) for 4 hours. (F) Cell viability on day 7 in PCa12 and PCa8, treated with various doses of ipatasertib, with the corresponding median inhibitory concentration (IC50) values displayed. (G) Cell viability on day 7 in PCa8 cells treated with the Akt inhibitor ipatasertib (500 nM), the MEK inhibitor PD0325901 (1 μM), the EGFR inhibitor gefi- tinib (1 μM), or DMSO. (H) The expression levels of downstream targets in the MAPK and PI3K pathways were assessed in PCa8 cells. Cells were treated with DMSO, the Akt inhibitor ipatasertib (500 nM), the MEK inhibitor PD0325901 (1 μM), and the EGFR inhibitor gefitinib (1 μM) for 4 hours [***P < 0.001 and ****P < 0.0001; (A and G) Welch’s t test; error bar represents ±SEM].

Article Snippet: The following antibodies were used for Western blotting: PSA (5365S), pAkt (Ser473) (Cell Signaling Technology, 4060L), pAkt (Thr308) (Cell Signaling Technology, 4056S), pEGFR (Cell Signaling Technology, 3777S), p- p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (Cell Signaling Technology, 4370S), pMEK1/2 (Ser217/221) (41G9) (Cell Signaling Technology, 9154S), pS6K (Thr389) (Cell Signaling Technology, 9205L), pS6 (Ser235/236) (Cell Signaling Technology, 4856S), PTEN (Cell Signaling Technology, 9188L), pPRAS40 (Thr246) (Cell Signaling Technology, 2997S), p4EBP1 (Thr37/46) (Cell Signaling Technology, 2855L), pmTOR (Ser2448) (Cell Signaling Technology, 5536S), TSC2 (Cell Signaling Technology, 4308S), HSP90 (Cell Signaling Technology, 4875S), GCN2 (Cell Signaling Technology, 3302S), pEIF2α (Ser51) (Cell Signaling Technology, 3398S), LC3A/B (Cell Signaling Technology, 12741S), LC3B (Cell Signaling Technology, 277S), β- actin (Cell Signaling Technology, 4970S), NPRL3 (Thermo Fisher, PA5- 52849), ATG4C (Cell Signaling Technology, 5262S), BIP (Cell Signaling Technology, 3177S), AR (Abcam, AB108341), FKBP5 (Cell Signaling Technology, 12210S), STEAP1 (Santa Cruz Biotechnology, SC- 271872), and PMEPA1 (Santa Cruz Biotechnology, SC- 293372).

Techniques: Inhibition, CRISPR, Modification, Expressing, Western Blot, Concentration Assay

Journal: Science advances

Article Title: Uncoupling of Akt and mTOR signaling drives resistance to Akt inhibition in PTEN loss prostate cancers.

doi: 10.1126/sciadv.adq3802

Figure Lengend Snippet: Fig. 4. Targeting mTOR directly within NPRL3 knockout contexts restores sensitivity to Akt inhibition. (A) Comparative growth of NPRL3-WT and NPRL3-KO LNCaP tumors in NSG mice subjected to ipatasertib treatment (50 mg/kg) or vehicle. Castration was performed when tumors reached ~300 mm3. (B) Western blot analysis illus- trating the status of PI3K signaling in LNCaP-sgNT and Lncap-sgNPRL3 tumors following a 4-week treatment period, as referenced in (A). (C) Immunohistochemistry im- ages illustrating the levels of pAkt473, pS6, p4EBP1, and Ki67 in LNCaP-sgNT and LNCaP-sgNPRL3 tumors following a 5-week treatment regimen. (D) Quantification of immunohistochemistry data of (C). (E) Cell viability analysis on day 7 in LNCaP-sgNT, LNCaP-sgNPRL3, Pca12-sgNT, and Pca12-sgNPRL3 cells treated with ipatasertib (500 nM), INK128 (100 nM), or DMSO. (F) LNCaP-sgNT and LNCaP-sgNPRL3 cells were treated with ipatasertib (500 nM), INK128 (100 nM), or DMSO for 4 hours. Cell lysates were subjected to Western blot analysis to assess protein expression levels in PI3K pathway {**P < 0.01, ***P < 0.001, and ****P < 0.0001; n.s., not significant; (A) multiple t test and [(D) and (E)] Welch’s t test; error bar represents ±SEM}.

Article Snippet: The following antibodies were used for Western blotting: PSA (5365S), pAkt (Ser473) (Cell Signaling Technology, 4060L), pAkt (Thr308) (Cell Signaling Technology, 4056S), pEGFR (Cell Signaling Technology, 3777S), p- p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (Cell Signaling Technology, 4370S), pMEK1/2 (Ser217/221) (41G9) (Cell Signaling Technology, 9154S), pS6K (Thr389) (Cell Signaling Technology, 9205L), pS6 (Ser235/236) (Cell Signaling Technology, 4856S), PTEN (Cell Signaling Technology, 9188L), pPRAS40 (Thr246) (Cell Signaling Technology, 2997S), p4EBP1 (Thr37/46) (Cell Signaling Technology, 2855L), pmTOR (Ser2448) (Cell Signaling Technology, 5536S), TSC2 (Cell Signaling Technology, 4308S), HSP90 (Cell Signaling Technology, 4875S), GCN2 (Cell Signaling Technology, 3302S), pEIF2α (Ser51) (Cell Signaling Technology, 3398S), LC3A/B (Cell Signaling Technology, 12741S), LC3B (Cell Signaling Technology, 277S), β- actin (Cell Signaling Technology, 4970S), NPRL3 (Thermo Fisher, PA5- 52849), ATG4C (Cell Signaling Technology, 5262S), BIP (Cell Signaling Technology, 3177S), AR (Abcam, AB108341), FKBP5 (Cell Signaling Technology, 12210S), STEAP1 (Santa Cruz Biotechnology, SC- 271872), and PMEPA1 (Santa Cruz Biotechnology, SC- 293372).

Techniques: Knock-Out, Inhibition, Western Blot, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a-b , Representative OCR ( a ) and ECAR ( b ) tracings of MC38 tumor cell fractions with indicated injections of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/AA). c-d Basal mitochondrial OCR ( c ) and cellular ECAR ( d ) of MC38 tumor fractions (n=5 mice). e , Unsupervised cluster analysis of differentially expressed metabolic mRNA transcripts of whole tumor, CD45-cancer cell, CD45+ CD11b+ F4/80hi TAM, CD45+ CD11b+ F4/80lo myeloid cell, CD45+ CD3+ CD8a+ T cell, and CD45+ CD3+ CD8a-(CD4+) T cell flow-sorted MC38 tumor populations (n=2 mice). f-g , Glucose transporter ( f ) and hexokinase ( g ) mRNA transcript levels of indicated MC38 tumor populations (n=2 mice). Dotted line approximates limit of detection. hi , Representative flow cytometry plots ( h ) and quantification ( i ) of pS6 levels in indicated MC38 tumor and spleen populations. j-k , Representative flow cytometry histograms ( j ) and quantification ( k ) of pS6 levels in cancer cells (CD45-CA9+), myeloid cells (CD45+ CD11b+ CD14+), T cells (CD45+ CD3+), and other immune cells (CD45+ CD3-CD14-) from patient ccRCC tumor and PBMC (n=4 patients). Each data point represents a biological replicate and error bars are SEM. a-d and f-g are representative of at least two independent experiments. P values were calculated using Welch’s 2-tailed t-test for (c-d) and Brown-Forsythe ANOVA test for (h, j). * p <0.05, ** p <0.01, *** p <0.001. ECAR: extracellular acidification rate; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; FMO: fluorescence minus one; MFI: median fluorescence intensity; OCR: oxygen consumption rate; PBMC: peripheral blood mononuclear cells; pS6: phosphorylated ribosomal protein S6 (Ser235/236).

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a , Sorting gates of MC38 tumor cells used for mRNA transcript analyses. b , Expression of selected cell identity markers in flow sorted MC38 tumor cell population. Dotted line approximates limit of detection. c-d , GLUT1 levels determined by flow cytometry in MC38 ( c ) and CT26 ( d ) tumor populations. e, Quantification of pS6 levels determined by flow cytometry in indicated CT26 tumor and spleen populations. Each data point represents a biological replicate and error bars are SEM. c-e are representative of independent experiments performed at least twice. P values were calculated using the Brown-Forsythe ANOVA test. *** p <0.001.

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Social boldness in male green anole lizards correlates with baseline vasopressin activity

doi: 10.1101/2021.09.11.459908

Figure Lengend Snippet: VP-immunoreactive cells (A) were scored as colocalized (white arrows) or not colocalized (yellow arrows) with pS6-immunoreactive signal (B). Nonspecific signal due to the presence of blood cells was disregarded because those cells would also cause autofluorescence within the tyrosine hydroxylase (TH) layer (C; captured for a separate study). A DAPI-stained layer was also captured (D).

Article Snippet: Briefly, we processed one series of brain sections with 1:250 dilution of rabbit anti-pS6 antibody (#2211, Cell Signaling Technology), 1:5000 dilution of guinea pig anti-vasopressin antibody (T-5048, Peninsula Laboratories), and 1:2000 dilution of sheep anti-tyrosine hydroxylase antibody (NB300-110, Novus Biologicals; part of a separate study).

Techniques: Staining

Journal: bioRxiv

Article Title: Social boldness in male green anole lizards correlates with baseline vasopressin activity

doi: 10.1101/2021.09.11.459908

Figure Lengend Snippet: Average boldness scores toward males within an agonistic context (A, B) but not females within a reproductive context (C, D) correlated negatively with the percentage of VP neurons in the PVN and SON that were pS6 positive.

Article Snippet: Briefly, we processed one series of brain sections with 1:250 dilution of rabbit anti-pS6 antibody (#2211, Cell Signaling Technology), 1:5000 dilution of guinea pig anti-vasopressin antibody (T-5048, Peninsula Laboratories), and 1:2000 dilution of sheep anti-tyrosine hydroxylase antibody (NB300-110, Novus Biologicals; part of a separate study).

Techniques: