Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: ( A ) N2a cells were treated with vehicle 1 (DMSO), vehicle 2 (ethanol), rapamycin (Rapa), PF-4708671 (PF), bisindolylmaleimide V (B5) or okadaic acid (OA) for 1 hour prior to cell lysis followed by immunoblotting for pFMRP, pS6K1, pS6, pERK T202/Y204 (readout for OA), and their total protein counterparts. Laddered bracket to right of tFMRP indicates FMRP isoforms and the asterisk denotes a nonspecific band. S6K1 isoforms are marked by p80 and p75. ( B and C ) Statistical verification of stable pFMRP:tFMRP across all conditions despite a significant decrease in pS6:S6 subsequent to mTORC1-S6K1 inhibition (B) and increase in pERK:ERK subsequent to PP2a inhibition (C). One way ANOVA with post-hoc Dunnett’s test (N = 4 per condition. Error bars = SEM). ( D ) Model of pathway and effect of pharmacological inhibitor. ( E ) N2a cells. Immunoblotting for pFMRP, tFMRP and mTORC1 pathway components from N2a cells maintained in 5% serum, transferred to increasing concentrations of serum (0, 2.5, 5 or 10%), and treated with vehicle (DMSO), rapamycin (Rapa) or PF-4708671 (PF) for 24 hours. Laddered bracket to the right of tFMRP blot indicates FMRP isoforms and the asterisk a nonspecific band. S6K1 isoforms are indicated by p85 and p70. The asterisk to the right of the total S6K1 blot indicates residual tFMRP signal from the blot above. ( F ) Bar graphs of (E). Statistical analysis: unmatched two-way ANOVA corrected for multiple comparisons with a post-hoc Tukey’s test. N = 4 per condition. Error bars = SEM.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Lysis, Western Blot, Inhibition

Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: ( A ) Cre expression and genetic recombination were verified by region-specific tdTomato expression (note tdTomato in telencephalon, Tel, but not cerebellum, Cb). Genotypes were confirmed by PCR: wild type mice (WT) have two wild type alleles (wt, 295bp amplicon), heterozygous mice (Het) have one wt and one mutant allele (mut, 370bp amplicon), and conditional mice have one floxed allele (fl, 480bp amplicon) with either a wt or mut allele. Asterisks indicate nonspecific bands ( B ) Immunoblotting of cortical lysates from P7 Tsc1 mice of different genotypes listed under the blots. ( C ) There is no statistical difference in pFMRP:tFMRP among genotypes despite significantly increased pS6:S6 in Tsc1 fl/mut vs. Tsc1 WT . pFMRP:tFMRP was quantified by unpaired, one-way ANOVA. pS6:S6 was quantified using unpaired, nonparametric one-sided Mann-Whitney test (** = p<0.01). Error bars STDEV. N = 3. ( D ) Model of serum-mediated mTORC1 pathway activation. ( E ) Immunoblots for pFMRP, pmTOR, pS6K1 and pS6 suggest that although the mTORC1 pathway responds to serum, pFMRP is not altered. N2a were cells maintained in 5% serum were transferred to 0, 2.5, 5 or 10% serum 1 hour prior to lysis. ( F ) Bar graphs of the phospho:total protein ratio shown in (E) showing that pFMRP:tFMRP is unchanged despite significantly increased mTORC1 pathway activity. Unpaired, nonparametric one-sided Mann-Whitney tests compared 0 and 10% serum conditions for each phospho-protein. We used a one-sided test because increased mTORC1 pathway activity is expected following serum application. Error bars = SEM. N = 4 per condition. ( G ) Model of mTORC1 pathway activation by overexpression. ( H ) Immunoblotting verified overexpression of transfected genes with the exception of TSC2 DN whose large C- and N-terminal deletions render it unrecognizable to many antibodies. N2a cell lysates were collected 48 hours post-transfection. ( I ) Immunoblotting for pFMRP, pS6K1, pS6 and their total protein counterparts. ( J ) Quantification of phospho:total protein ratios normalized to mock transfected cells – unpaired, one-way ANOVA corrected for multiple comparisons. P values were derived from post-hoc Dunnett’s test (*, **, *** & **** = P≤0.05, 0.01, 0.001 and 0.0001 respectively). N = 4 per condition.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Expressing, Amplification, Mutagenesis, Western Blot, MANN-WHITNEY, Activation Assay, Lysis, Activity Assay, Over Expression, Transfection, Derivative Assay

Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: ( A ) CD1 mice were intraperitoneally injected (IP) with PF (75 mg/kg) and sacrificed at various time points thereafter. Hyperphosphorylation of S6K1 T389 (pS6K1) detected at 2 hours suggests that this compound can cross the blood brain barrier. N = 3 per time point. ( B ) Immunoblots from cortical lysates from CD1 mice treated IP with rapamycin (Rapa, 1.5 mg/kg for 5 days), PF-4708671 (PF, 75 mg/kg for 2 hours), and vehicle (DMSO) alone. Asterisks indicate nonspecific bands, the arrow indicates the pFMRP isoform, laddered bracket indicates the tFMRP isoforms, and S6K1 isoforms are indicated by p85 and p70. ( C ) Quantification verifies a significant decrease in pS6:S6 but no change in pFMRP:FMRP following mTORC1 or S6K1 inhibition in vivo . * P <0.05 and ** P <0.01 by unpaired, one-sided Mann-Whitney Test. A one-sided test was used considering that decreased pS6 levels were expected. N = 6 per condition. Error bars = SEM.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Injection, Western Blot, Inhibition, In Vivo, MANN-WHITNEY

Journal: PLoS ONE

Article Title: FMRP S499 Is Phosphorylated Independent of mTORC1-S6K1 Activity

doi: 10.1371/journal.pone.0096956

Figure Lengend Snippet: List of antibodies.

Article Snippet: pS6 S240/244 , Cell Signal (Rb) #5364 , 1∶10,000;20,000 , 5% BSA/TBST (total S6 must be probedfor first as pS6 is not efficiently stripped).

Techniques: Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Effects of Early Exposure to Isoflurane on Susceptibility to Chronic Pain Are Mediated by Increased Neural Activity Due to Actions of the Mammalian Target of the Rapamycin Pathway

doi: 10.3390/ijms241813760

Figure Lengend Snippet: Effect of isoflurane exposure on neuronal activity in superficial DSC. ( A ) The photographs show pS6 (red channel) double-immunolabeled with NeuN (green) in DSC neurons (lamina I-II) in each group. ( B ) The histogram showed statistical results ( n = 6). Compared to naïve group, Ctrl+SNI+Veh group showed a trend of increased number of pS6 positive neurons. Iso+SNI+Veh group showed significantly increased pS6+/NeuN+ cells, as compared to Ctrl+SNI+Veh group and naïve group. Rapa treatment reduced this change. Bar = 50 μm. One-way ANOVA for statistics. ns: no significance; **: p < 0.01; ***: p < 0.001; ****: 0.0001.

Article Snippet: The following primary antibodies were used for double or single immunolabeling: (1) rabbit anti-phospho-S6 (pS6) (1:1000; Cell Signaling, Danvers, MA, USA; Cat#: 2211); (2) mouse anti-c-fos (1:1000; Abcam; Cambridge, MA, USA; Cat#: ab298942); (3) mouse anti-NeuN (1: 200; EDM Millipore; Darmstadt, Germany; Cat#: MAB377); (4) rabbit anti-NeuN (1:200; EDM Millipore; Darmstadt, Germany; Cat#: ABN78); (5) mouse anti GFAP (1:1000; Millipore-Sigma; Burlington, MA, USA; Cat#: MAB360); (6) rabbit anti-BrdU (1:500; Rockland; Limerick, PA, USA; Cat#: 660-410-C29); (7) rabbit anit-Iba1 (1:1000; Wako; Osaka, Japan; Cat#: 019-19741); (8) mouse anti-p-P38 MAPK (1:200; Cell Signaling, Danvers, MA, USA; Cat#: #9216); (9) Mouse anti-CGRP (1:1;000; Abcam; Cambridge, MA, USA; Cat#: ab81887); (10) Rabbit anti-connexin-43 (Cx43) (1:500; Sigma-Aldrich; St. Louis, MO, USA; Cat#: C6219).

Techniques: Activity Assay, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: Effects of Early Exposure to Isoflurane on Susceptibility to Chronic Pain Are Mediated by Increased Neural Activity Due to Actions of the Mammalian Target of the Rapamycin Pathway

doi: 10.3390/ijms241813760

Figure Lengend Snippet: WB study shows alteration of neural activity markers in DSC. ( A ) The ratio of pS6 band intensity over β-actin in naïve and mice with SNI-produced chronic pain was at a same level. The amount in Iso+SNI+Veh group was dramatically higher than in Ctrl+SNI+Veh group and was attenuated by Rapa treatment close to naïve. ( B ) The intensity pattern of c-fos was similar to pS6. Numbers for naïve and Ctrl+SNI+Veh groups were identical. Iso+SNI+Veh enhanced, and Rapa reversed the reactive intensity. ( C ) Ctrl+SNI+Veh did not, but Iso+SNI+Veh did elevate molecule level of N-cadherin. Rapamycin restored this level to Ctrl+SNI+Veh animals. ( D ) The ratio of p-CREB over GAPDH, another standard marker, in naïve and Ctrl+SNI+Veh was almost same, but the amount in Iso+SNI+Veh was significantly higher. Rapa attenuated the level of p-CREB, and this number was near naïve. ( E ) The ratio of GFAP band intensity over GAPDH in Ctrl+SNI+Veh was elevated compared to naïve. This number was raised by Iso+SNI+Veh and a significant recovery resulted from Rapa treatment. ( F ) The mRNA of P2Y12 in DSC was examined with qPCR. Ctrl+SNI+Veh partially enhanced the relative level of P2Y12 than naive and Iso+SNI+Veh obviously increased P2Y12 than SNI only. This number was reversed by Rapa treatment close to naive. One-way ANOVA for all figures ( n = 6 for A, B, C, D, E; n = 4 for F). ns: no significance; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: The following primary antibodies were used for double or single immunolabeling: (1) rabbit anti-phospho-S6 (pS6) (1:1000; Cell Signaling, Danvers, MA, USA; Cat#: 2211); (2) mouse anti-c-fos (1:1000; Abcam; Cambridge, MA, USA; Cat#: ab298942); (3) mouse anti-NeuN (1: 200; EDM Millipore; Darmstadt, Germany; Cat#: MAB377); (4) rabbit anti-NeuN (1:200; EDM Millipore; Darmstadt, Germany; Cat#: ABN78); (5) mouse anti GFAP (1:1000; Millipore-Sigma; Burlington, MA, USA; Cat#: MAB360); (6) rabbit anti-BrdU (1:500; Rockland; Limerick, PA, USA; Cat#: 660-410-C29); (7) rabbit anit-Iba1 (1:1000; Wako; Osaka, Japan; Cat#: 019-19741); (8) mouse anti-p-P38 MAPK (1:200; Cell Signaling, Danvers, MA, USA; Cat#: #9216); (9) Mouse anti-CGRP (1:1;000; Abcam; Cambridge, MA, USA; Cat#: ab81887); (10) Rabbit anti-connexin-43 (Cx43) (1:500; Sigma-Aldrich; St. Louis, MO, USA; Cat#: C6219).

Techniques: Activity Assay, Produced, Marker

Journal: International Journal of Molecular Sciences

Article Title: Effects of Early Exposure to Isoflurane on Susceptibility to Chronic Pain Are Mediated by Increased Neural Activity Due to Actions of the Mammalian Target of the Rapamycin Pathway

doi: 10.3390/ijms241813760

Figure Lengend Snippet: Effect of early isoflurane exposure on DRG neurons with IHC. ( A ) Effect of isoflurane and SNI on neuronal activity in DRG. In the chronic pain model (Ctrl+SNI+Veh), the number of pS6+ over all neurons was higher than in naive, but not statistically significant. Isoflurane obviously increased and rapamycin dramatically decreased this number, and it was even lower than naïve. ( B ) In naïve, CGRP was mainly expressed in small-sized (<25 µm) neurons. In Ctrl+SNI+Veh mice, the percentage of CGRP+ over total neurons was identical with naïve. In the Iso+SNI+Veh group, this number was increased and certain number of medium sized CGRP+ neurons (>25 µm) appeared. Rapa restored the percentage of CGRP+ neurons near naive. Bars = 25 µm. One-way ANOVA ( n = 6). ns: no significance; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: The following primary antibodies were used for double or single immunolabeling: (1) rabbit anti-phospho-S6 (pS6) (1:1000; Cell Signaling, Danvers, MA, USA; Cat#: 2211); (2) mouse anti-c-fos (1:1000; Abcam; Cambridge, MA, USA; Cat#: ab298942); (3) mouse anti-NeuN (1: 200; EDM Millipore; Darmstadt, Germany; Cat#: MAB377); (4) rabbit anti-NeuN (1:200; EDM Millipore; Darmstadt, Germany; Cat#: ABN78); (5) mouse anti GFAP (1:1000; Millipore-Sigma; Burlington, MA, USA; Cat#: MAB360); (6) rabbit anti-BrdU (1:500; Rockland; Limerick, PA, USA; Cat#: 660-410-C29); (7) rabbit anit-Iba1 (1:1000; Wako; Osaka, Japan; Cat#: 019-19741); (8) mouse anti-p-P38 MAPK (1:200; Cell Signaling, Danvers, MA, USA; Cat#: #9216); (9) Mouse anti-CGRP (1:1;000; Abcam; Cambridge, MA, USA; Cat#: ab81887); (10) Rabbit anti-connexin-43 (Cx43) (1:500; Sigma-Aldrich; St. Louis, MO, USA; Cat#: C6219).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Effects of Early Exposure to Isoflurane on Susceptibility to Chronic Pain Are Mediated by Increased Neural Activity Due to Actions of the Mammalian Target of the Rapamycin Pathway

doi: 10.3390/ijms241813760

Figure Lengend Snippet: WB data show the effect of isoflurane exposure on mTOR expression and neural activity in DRG. The ratio of detected molecule density over standard markers, β-actin or GAPDH, was calculated. ( A ) Ctrl+SNI+Veh increased pS6 level to higher than naive, but the difference was not significant. Iso+SNI+Veh elevated this number by a large margin and Rapa reduced the pS6 intensity. ( B ) A neuronal activity marker, p-ERK, was used to detect neuronal activity. Ctrl+SNI+Veh increased p-ERK level to higher than naïve and Iso+SNI+Veh produced a huge increase. Rapa reduced the p-ERK react density. ( C ) GFAP was used to evaluate the level of SGCs. Ctrl+SNI+Veh increased GFAP level than naïve and Iso+SNI+Veh further upregulated the intensity than Ctrl+SNI+Veh. This number was reversed with Rapa treatment. ( D ) Ctrl+SNI+Veh did not, but Iso+SNI+Veh did, significantly increase Iba1 level to higher than naïve. Iso+SNI+Rapa downregulated iba1 reactivity close to naive. One-way ANOVA ( n = 6). ns: no significance; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The following primary antibodies were used for double or single immunolabeling: (1) rabbit anti-phospho-S6 (pS6) (1:1000; Cell Signaling, Danvers, MA, USA; Cat#: 2211); (2) mouse anti-c-fos (1:1000; Abcam; Cambridge, MA, USA; Cat#: ab298942); (3) mouse anti-NeuN (1: 200; EDM Millipore; Darmstadt, Germany; Cat#: MAB377); (4) rabbit anti-NeuN (1:200; EDM Millipore; Darmstadt, Germany; Cat#: ABN78); (5) mouse anti GFAP (1:1000; Millipore-Sigma; Burlington, MA, USA; Cat#: MAB360); (6) rabbit anti-BrdU (1:500; Rockland; Limerick, PA, USA; Cat#: 660-410-C29); (7) rabbit anit-Iba1 (1:1000; Wako; Osaka, Japan; Cat#: 019-19741); (8) mouse anti-p-P38 MAPK (1:200; Cell Signaling, Danvers, MA, USA; Cat#: #9216); (9) Mouse anti-CGRP (1:1;000; Abcam; Cambridge, MA, USA; Cat#: ab81887); (10) Rabbit anti-connexin-43 (Cx43) (1:500; Sigma-Aldrich; St. Louis, MO, USA; Cat#: C6219).

Techniques: Expressing, Activity Assay, Marker, Produced

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