Journal: bioRxiv

Article Title: MYC disrupts transcriptional and metabolic circadian oscillations in cancer and promotes enhanced biosynthesis

doi: 10.1101/2023.01.03.522637

Figure Lengend Snippet: A-C. U2OS MYC-ER ( A ), SHEP N-MYC-ER, ( B ), and SKNAS N-MYC-ER ( C ) were treated with ethanol control (MYC-OFF) or 4-hydroxytamoxifen (MYC-ON) (4OHT) to activate MYC, and entrained with dexamethasone, and after 24 hours, protein was collected every 4 hours for the indicated time period. Protein lysates were prepared to preserve protein glycosylation (see Methods), and immunoblot was performed for the indicated proteins. For some targets [4F2hc, LAT1, REV-ERBα (abbreviated REVα)], a darker exposure (‘dark’) and lighter exposure (‘light’) of the same blot are presented. For GLUT1, # indicates a non-specific band. Some samples (CT26 for U2OS, CT32 for SHEP and SKNAS) were treated with PNGase-F prior to immunoblot to remove glycosylation marks. Note that the PNGase-F lanes have less protein loaded than the other lanes. Data represent n=2 biological replicates for U2OS and one each for SHEP and SKNAS. D. On-cell western of U2OS MYC-ER cells ± MYC for LAT1. U2OS MYC-ER cells were grown on a 24-well tissue culture plate ± 4OHT for 48 hours, fixed with formaldehyde but not permeabilized, and then stained with the indicated antibody or IgG control. CellStain 700 indicates cell density in each well. Data represent at least two independent experiments of 6 biological replicate wells each. E. Quantitation of LAT1 or IgG from ( D ). * indicates P < 0.00001 by Welch’s Corrected Student’s T-test. F. LC-Mass spectrometry was performed on U2OS MYC-ER treated ± 4OHT for at least 48 hours. N=25 circadian timepoints for MYC-OFF and MYC-ON were averaged as biological replicates, normalized to cell number for each collection. * indicates p < 0.05 by Welch’s Corrected Student’s T-test.

Article Snippet: Wells were washed with tris-buffered saline, and stained with primary antibodies mouse anti-LAT1 BU53 (Novus NBP2-50465AF647) or mouse IgG2A isotype control (Novus IC003R), and secondary antibodies goat anti-mouse Alexa Flour 790 (Thermo Scientific A11357) or CellTag 700 Stain (Licor 926-41090), which is used to quantify total cell number and intensity.

Techniques: Control, Western Blot, Staining, Quantitation Assay, Mass Spectrometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Metformin and 2-Deoxyglucose Collaboratively Suppress Human CD4 + T Cell Effector Functions and Activation-Induced Metabolic Reprogramming.

doi: 10.4049/jimmunol.2000137

Figure Lengend Snippet: FIGURE 4. Metformin + 2-DG represses MYC-dependent glutamine uptake and catabolism. (A) RNA-Seq results for MYC-dependent genes involved in glutamine metabolism in primary human CD4+ T cells activated with anti-CD3/anti-CD28 for 24 h in the presence of metformin + 2-DG or vehicle alone. Each tile in the heatmap represents average log transcripts per million (TPM) from triplicates of one of three human donors. (B) Time course measurements of 14C-glutamine uptake into human CD4+ T cells activated for 24 h with anti-CD3/anti-CD28 in the presence of metformin + 2-DG or vehicle alone. (C and D) Flow cytometry analysis of mean fluorescence intensity of (C) SLC7A5 and (D) CD98 on human CD4+ T cells activated for 24 h with anti-CD3/anti- CD28 in the presence of metformin + 2-DG or vehicle alone, and representative flow cytometry plots showing percentage of (C) SLC7A5- and (D) CD98- positive cells. (E) Time course measurements of 14C-leucine uptake into human CD4+ T cells activated for 24 h with anti-CD3/anti-CD28 in the presence of metformin + 2-DG or vehicle alone. All data shown are the mean 6 SEM of three to six technical replicates. All data shown are representative of at least two independent experiments with at least two different human donors. ***p , 0.001, ****p , 0.0001.

Article Snippet: Abs against SLC7A5 were from Novus Biologicals (catalog no. NBP2-50465AF647).

Techniques: RNA Sequencing, Flow Cytometry, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Metformin and 2-Deoxyglucose Collaboratively Suppress Human CD4 + T Cell Effector Functions and Activation-Induced Metabolic Reprogramming.

doi: 10.4049/jimmunol.2000137

Figure Lengend Snippet: FIGURE 5. Deletion of MYC in primary human T cells impairs proliferation and glutamine uptake. (A) Immunoblot confirming loss of MYC (left) and HIF-1A (right) protein in human MYC KO and HIF-1A KO CD4+ T cells activated for 24 h with anti-CD3/anti-CD28. (B) Cell proliferation measured by dilution of CTV dye. Representative CTV dilution plot and proliferative index of human MYC KO, HIF-1A KO, and MYC/HIF-1A dKO CD4+ T cells following 96 h of activation with anti-CD3/anti-CD28. (C and D) Flow cytometry analysis of (C) CD98 and (D) SLC7A5 surface expression on MYC KO, HIF-1A KO, and MYC/HIF-1A dKO CD4+ T cells activated for 24 h with anti-CD3/anti-CD28. Mean fluorescence intensity (MFI) shown are the mean 6 SEM (n = 3–6). *p , 0.05, **p , 0.01. (E) 14C-glutamine uptake allowed to proceed for 1 h in MYC KO, HIF-1A KO, and MYC/HIF-1A dKO CD4+

Article Snippet: Abs against SLC7A5 were from Novus Biologicals (catalog no. NBP2-50465AF647).

Techniques: Western Blot, Activation Assay, Flow Cytometry, Expressing

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A ) Schematic diagram of the procedure for [ 125 I]IMT uptake assay. ( B and C ) Brain tissues from wild-type mice at 8–12 weeks of age were incubated with [ 125 I]IMT at 4°C or 37°C for 30 minutes in Hanks’ balanced salt solution (HBSS) buffer ( B ) ( n = 5, *** P < 0.001, 2-tailed Student’s t test) or at 37°C for 30 minutes in HBSS buffer containing 30 μM JPH203 ( C ) ( n = 10 or 11, *** P < 0.001, 2-tailed Student’s t test). ( D and E ) Quantification of mRNAs encoding LAT family in VMH ( D ) and ARC ( E ) of mice fed a normal chow diet (NC) at 8–12 weeks of age ( n = 4, * P < 0.05, ** P < 0.01, Kruskal-Wallis test post hoc Dunn’s test). TPM, transcripts per million. ( F ) Quantification of mRNAs encoding LAT family in human hypothalamus ( n = 202, *** P < 0.001, Kruskal-Wallis test post hoc Dunn’s test). ( G ) Brain tissues from WT mice fed an NC or HFD (for 17 weeks, from 7 to 24 weeks of age) at 24 weeks of age were incubated with [ 125 I]IMT at 37°C for 30 minutes in HBSS buffer ( n = 5, * P < 0.05, 2-tailed Student’s t test). ( H ) Brain tissues from WT mice and db/db mice at 24 weeks of age were incubated with [ 125 I]IMT at 37°C for 30 minutes in HBSS buffer ( n = 5, *** P < 0.001, 2-tailed Student’s t test). ( I ) LAT1-dependent [ 125 I]IMT uptake in brain tissues from WT mice and db/db mice at 24 weeks of age ( n = 5, ** P < 0.01, 2-tailed Student’s t test). ( J and K ) Quantification of Slc7a5 mRNA in VMH ( J ) and ARC ( K ) of mice fed an NC or HFD (from 5–6 to 8–12 weeks of age) at 8–12 weeks of age ( n = 4, * P < 0.05, *** P < 0.001, Fisher’s exact test). All the mice used in this study were male. CPM, counts per million.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Incubation

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A ) Weekly body weight is shown for LepR-Cre Slc7a5 fl/fl mice and control mice fed an NC ( n = 8 or 9, * P < 0.05, ** P < 0.01, *** P < 0.001, 2-way ANOVA with Bonferroni post hoc test). ( B ) Gross appearance at 24 weeks of age is shown for LepR-Cre Slc7a5 fl/fl mice and control mice fed an NC. Scale bar, 1 cm. ( C – E ) Representative pictures of visceral and subcutaneous WAT (vWAT and sWAT) ( C ), adipose tissue weights ( D ), and adipose tissue weights normalized to body weight ( E ) are shown for LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age ( n = 17 or 18, ** P < 0.01, *** P < 0.001, 2-tailed Student’s t test). Scale bar, 1 cm. ( F and G ) H&E stain was performed on the vWAT of LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age, followed by quantification of adipocyte size of vWAT ( n = 15 or 16, * P < 0.05, ** P < 0.01, *** P < 0.001, 2-way ANOVA with Bonferroni post hoc test). Scale bar, 100 μm. ( H ) Serum leptin levels were measured in LepR-Cre Slc7a5 fl/fl mice at 22–24 weeks of age ( n = 4, *** P < 0.001, 2-tailed Student’s t test). ( I ) [ 125 I]IMT uptake in hypothalamus from LepR-Cre Slc7a5 fl/fl mice at 22–24 weeks of age ( n = 6 or 7, * P < 0.05, 2-tailed Student’s t test). ( J ) Log 2 ratio of the amino acid levels in VMH between LepR-Cre Slc7a5 fl/fl mice and control mice at 7 weeks of age ( n = 12, the amino acids with P < 0.05 are represented in red, 2-tailed Student’s t test). ( K ) Quantification of Slc7a5 mRNA in ARC and VMH of mice fed an NC ( n = 4, ** P < 0.01, Fisher’s exact test). ( L and M ) The enrichment plots for amino acid transport-related gene sets in VMH and ARC of mice fed an NC ( n = 4). All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Control, Staining

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A ) Glucose tolerance tests (GTTs) were performed in LepR-Cre Slc7a5 fl/fl mice and control mice after a 6-hour fast at 22–24 weeks of age ( n = 6 to 8, 2-way ANOVA with Bonferroni post hoc test). ( B ) Insulin tolerance tests (ITTs) were performed in LepR-Cre Slc7a5 fl/fl mice and control mice after a 6-hour fast at 22–24 weeks of age ( n = 10 or 11, * P < 0.05, ** P < 0.01, *** P < 0.001, 2-way ANOVA with Bonferroni post hoc test). ( C ) Serum insulin levels were measured in LepR-Cre Slc7a5 fl/fl mice and control mice after a 6-hour fast at 22–24 weeks of age ( n = 5, *** P < 0.001, 2-tailed Student’s t test). ( D and E ) Immunohistochemical stain for insulin was performed on the islets of LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age, followed by quantification of insulin-positive area ( n = 6 to 10, * P < 0.05, 2-tailed Student’s t test). Scale bar, 200 μm. ( F – H ) Representative picture of liver ( F ), tissue weights ( G ), and tissue weights normalized to body weight ( H ) are shown for LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age ( n = 17 or 18, *** P < 0.001, 2-tailed Student’s t test). Scale bar, 500 μm. ( I ) Oil Red O stain was performed on the liver of LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age. Scale bar, 100 μm. All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Control, Immunohistochemical staining, Staining

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A – F ) Food intake ( A ), locomotor activity ( B ), O 2 consumption ( C ), CO 2 production ( D ), energy expenditure ( E ), and body temperature ( F ) were measured in singly housed LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age ( n = 7 to 10, * P < 0.05, ** P < 0.01, *** P < 0.001, 2-tailed Student’s t test). ( G – I ) Representative picture of BAT ( G ), tissue weights ( H ), and tissue weights normalized to body weight ( I ) are shown for LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age ( n = 17 or 18, ** P < 0.01, *** P < 0.001, 2-tailed Student’s t test). Scale bar, 1 cm. ( J ) H&E stain was performed on the BAT of LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age. Scale bar, 100 μm. ( K ) Transmission electron microscopy analysis was performed on the BAT of LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age. Scale bar, 10 μm. ( L and M ) mtDNA content of BAT ( L ) and mRNA expression ( M ) were measured in LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age ( n = 3 to 4, * P < 0.05, ** P < 0.01, *** P < 0.001, 2-tailed Student’s t test). All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Activity Assay, Control, Staining, Transmission Assay, Electron Microscopy, Expressing

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A and B ) Serum epinephrine levels were measured in LepR-Cre Slc7a5 fl/fl mice and control mice at 7 ( A ) or 24 ( B ) weeks of age (7 wk; n = 8, 24 wk; n = 4, ** P < 0.01, 2-tailed Student’s t test). ( C and D ) Serum norepinephrine levels were measured in LepR-Cre Slc7a5 fl/fl mice and control mice at 7 ( C ) or 24 ( D ) weeks of age (7 wk; n = 8, 24 wk; n = 4, *** P < 0.001, 2-tailed Student’s t test). ( E and F ) Urine epinephrine levels were measured in LepR-Cre Slc7a5 fl/fl mice and control mice at 7 ( E ) or 24 ( F ) weeks of age (7 wk; n = 8, 24 wk; n = 4, ** P < 0.01, 2-tailed Student’s t test). ( G and H ) Urine norepinephrine levels were measured in LepR-Cre Slc7a5 fl/fl mice and control mice at 7 ( G ) or 24 ( H ) weeks of age (7 wk; n = 8, 24 wk; n = 4, * P < 0.05, 2-tailed Student’s t test). ( I and J ) Norepinephrine turnover levels of ( I ) BAT and ( J ) soleus muscle were measured in LepR-Cre Slc7a5 fl/fl mice and control mice at 24 weeks of age ( n = 8 to 10, ** P < 0.01, *** P < 0.001, 2-tailed Student’s t test). ( K ) Serum leptin levels were measured in LepR-Cre Slc7a5 fl/fl mice at 7 weeks of age ( n = 8, ** P < 0.01, 2-tailed Student’s t test). ( L – O ) Immunohistochemical analysis of phosphorylated STAT3 activation 2 hours after intraperitoneal injection of 5 mg/kg leptin was performed on the VMH ( L and M ) and ARC ( N and O ) of LepR-Cre Slc7a5 fl/fl mice and control mice at 7 weeks of age ( n = 3 or 4, * P < 0.05, ** P < 0.01, # P < 0.05, 2-way ANOVA with Bonferroni post hoc test). Scale bar, 100 μm. Arrowheads indicate representative phosphorylated STAT3/tdTomato double-positive neurons. All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Control, Immunohistochemical staining, Activation Assay, Injection

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A ) Micro–computed tomography (μCT) analysis (scale bar, 1 mm); ( B ) bone volume over tissue volume (BV/TV) ratio as determined by μCT ( n = 12 or 13, * P < 0.05, 2-tailed Student’s t test); ( C ) von Kossa stain, scale bar, 1 mm; ( D ) BV/TV ratio as determined by von Kossa stain ( n = 5 or 6, * P < 0.05, 2-tailed Student’s t test); ( E ) number of osteoblasts/tissue area ratio ( n = 5 or 6, ** P < 0.01, 2-tailed Student’s t test); ( F ) calcein labeling (scale bar, 50 μm); ( G ) bone formation rate ( n = 5 or 6, * P < 0.05, 2-tailed Student’s t test); ( H ) TRAP stain (scale bar, 50 μm; arrowheads indicate TRAP-positive osteoclasts); and ( I ) osteoclast surface/bone surface ratio of femurs from LepR-Cre Slc7a5 fl/fl mice and control mice at 12–16 weeks of age ( n = 3 to 5, ** P < 0.01, 2-tailed Student’s t test). TRAP, tartrate-resistant acid phosphatase. ( J ) μCT analysis, scale bar, 1 mm and ( K ) BV/TV ratio as determined by μCT of femurs from LepR-Cre Slc7a5 fl/fl mice administrated with isoproterenol at 14 weeks of age ( n = 10, ** P < 0.01, # P < 0.05, 2-tailed Student’s t test with Benjamini-Hochberg correction). ( L ) Representative images of CFU assays stained with crystal violet and alizarin red (scale bar, 500 μm), ( M ) mRNA level of Slc7a5 in BM-MSCs ( n = 4, ** P < 0.01, 2-tailed Student’s t test), and ( N and O ) quantification of CFU assays stained with crystal violet ( N ) and alizarin red ( O ) ( n = 6 to 9, ** P < 0.01, *** P < 0.001, 2-tailed Student’s t test) in LepR-Cre Slc7a5 fl/fl mice at 6 weeks of age. All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Micro-CT, Staining, Labeling, Control

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A ) Schematic diagram of the bilateral viral microinjection into the VMH and representative validation image of mCherry expression in the VMH. Scale bar, 500 μm. ( B ) Weekly body weight after injection of AAV- Control or AAV- Slc7a5 into the VMH is shown for LepR-Cre Slc7a5 fl/fl mice and control mice ( n = 8 to 12, ** P < 0.01, *** P < 0.001: versus LepR-Cre /AAV- Control , # P < 0.05, ## P < 0.01, ### P < 0.001: versus LepR-Cre Slc7a5 fl/fl /AAV- Control , 2-way ANOVA with Bonferroni post hoc test). ( C and D ) Adipose tissue weights ( C ) and adipose tissue weights normalized to body weight ( D ) are shown for LepR-Cre Slc7a5 fl/fl mice and control mice injected with AAV- Control or AAV- Slc7a5 into the VMH at 16 weeks of age ( n = 8, ** P < 0.01, *** P < 0.001, # P < 0.05, ## P < 0.01, ### P < 0.001, 2-tailed Student’s t test with Bonferroni correction). ( E ) ITTs were performed in LepR-Cre Slc7a5 fl/fl mice and control mice injected with AAV- Control or AAV- Slc7a5 into the VMH after a 6-hour fast at 16 weeks of age ( n = 3 or 4, * P < 0.05, ** P < 0.01: versus LepR-Cre /AAV- Control , # P < 0.05, ## P < 0.01, ### P < 0.001: versus LepR-Cre Slc7a5 fl/fl /AAV- Control , 2-way ANOVA with Bonferroni post hoc test). ( F ) μCT analysis (scale bar, 1 mm) and ( G ) BV/TV ratio as determined by μCT of femurs from LepR-Cre Slc7a5 fl/fl mice and control mice injected with AAV- Control or AAV- Slc7a5 into the VMH at 12–16 weeks of age ( n = 6 to 11, * P < 0.05, # P < 0.05, 2-way ANOVA with Bonferroni post hoc test). All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Microinjection, Expressing, Injection, Control

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A and B ) Immunohistochemical analysis of pS6 activation 2 hours after intraperitoneal injection of 5 mg/kg leptin was performed on the VMH of LepR-Cre Slc7a5 fl/fl mice and control mice at 7 weeks of age ( n = 3, ** P < 0.01, # P < 0.05, 2-way ANOVA with Bonferroni post hoc test). Scale bar, 100 μm. Arrowheads indicate pS6/tdTomato double-positive neurons. ( C ) Gross appearance at 24 weeks of age is shown for LepR-Cre Slc7a5 fl/fl mice, LepR-Cre Slc7a5 fl/fl Tsc1 fl/+ mice, and control mice fed an NC. Scale bar, 1 cm. ( D ) Weekly body weight is shown for LepR-Cre Slc7a5 fl/fl mice, LepR-Cre Slc7a5 fl/fl Tsc1 fl/+ mice, and control mice fed NC ( n = 9 to 11, * P < 0.05, ** P < 0.01, *** P < 0.001: versus Slc7a5 fl/fl , # P < 0.05, ## P < 0.01, ### P < 0.001: versus LepR-Cre Slc7a5 fl/fl , 2-way ANOVA with Bonferroni post hoc test). ( E – G ) Representative pictures of vWAT, sWAT, and BAT ( E ); adipose tissue weights ( F ); and adipose tissue weights normalized to body weight ( G ) are shown for LepR-Cre Slc7a5 fl/fl mice and control mice at 22–24 weeks of age ( n = 12 to 18, ** P < 0.01, *** P < 0.001, ### P < 0.001, 1-way ANOVA with Bonferroni post hoc test). Scale bar, 1 cm. ( H ) ITTs were performed in LepR-Cre Slc7a5 fl/fl mice, LepR-Cre Slc7a5 fl/fl Tsc1 fl/+ mice, and control mice after a 6-hour fast at 22–24 weeks of age ( n = 3 to 9, * P < 0.05, ** P < 0.01: versus Slc7a5 fl/fl , ## P < 0.01, ### P < 0.001: versus LepR-Cre Slc7a5 fl/fl , 2-way ANOVA with Bonferroni post hoc test). All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Immunohistochemical staining, Activation Assay, Injection, Control

Journal: JCI Insight

Article Title: l- Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis

doi: 10.1172/jci.insight.154925

Figure Lengend Snippet: ( A ) μCT analysis, scale bar, 1 mm; and ( B ) BV/TV ratio as determined by μCT of femurs from LepR-Cre Slc7a5 fl/fl mice, LepR-Cre Slc7a5 fl/fl Tsc1 fl/+ mice, and control mice at 12–16 weeks of age ( n = 3 to 14, *** P < 0.001, # P < 0.05, 1-way ANOVA with Bonferroni post hoc test). ( C ) Working model of the findings of this study. All the mice used in this study were male.

Article Snippet: pAAV - hSynI - DIO - HA - Slc7a5 - mCherry vector was generated by replacing rM3D(Gs) cDNA in pAAV-hSyn-DIO-rM3D(Gs)-mCherry (Addgene 50458, a gift from Bryan Roth, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA) with a mouse Slc7a5 cDNA from pCMV6 - Slc7a5 (Origene, MC217270) vector. pAAV - hSynI - DIO - HA - mCherry vector was generated by deleting rM3D(Gs) cDNA from pAAV-hSyn-DIO-rM3D(Gs)-mCherry .

Techniques: Control

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

doi: 10.1128/jvi.01146-08

Figure Lengend Snippet: FIG. 3. KSHV interactions with integrins and CD98/xCT molecules. (A, B, C, and D) Serum- starved HMVEC-d were uninfected (UN) or infected with KSHV at an MOI of 10 DNA copies/cell for different time periods (minutes). Infected and uninfected cells were lysed with NP-40-containing buffer, and the protein complexes were immunoprecipitated. (A) CD98hc coimmunoprecipitates with V5 and V3 integrins in KSHV-infected HMVEC-d. Shown is immunoprecipitation (IP) with anti-V5 integrin antibody (top) or anti-V3 integrin antibodies (middle). The immunoprecipitated proteins were Western blotted and probed with anti-CD98hc antibodies. The immunoprecipitated blot was stripped and reprobed with anti-3 integrin antibody (bottom). (B) CD98hc coimmunoprecipitates with 1 and 31 integrins in KSHV-infected HMVEC-d. Shown is IP with anti-1 integrin (top) or anti-31 integrin antibody (middle). The immunoprecipitates were Western blotted and probed for CD98hc. Equal quantities of total cell lysates were probed with anti-1 antibody (bottom). (C) xCT coimmunoprecipitates with V5 and V3 integrins in KSHV-infected HMVEC-d. Infected and uninfected cell lysates were immunoprecipitated with anti-V5 integrin antibody (top) or anti-V3 integrin antibody (middle). The immunoprecipitated proteins were Western blotted and probed with xCT antibodies. The blot was stripped and reprobed with anti-3 integrin antibody (bottom). (D) xCT coimmunoprecipitates with 1 and 31 integrins in KSHV-infected cells. Shown is IP with 1 (top) or 31 (middle) integrin antibody. The immunoprecipitates were blotted and probed with anti-xCT antibody. The immunoprecipitated blot was stripped and reprobed with anti-3 antibody (bottom). (E) Western blot analysis of xCT expression in HMVEC-d. Equal quantities of total cell lysates of the samples in Fig. 1D were subjected to SDS- polyacrylamide gel electrophoresis followed by blotting with anti xCT antibody. (F) There is no association between 31 integrin and LAT1 in KSHV-infected cells. Infected and uninfected HMVEC-d were lysed, and the lysates were immunoprecipitated using specific anti-31 integrin antibodies. The immunoprecipitated proteins were blotted and probed with anti-LAT1 antibodies (top). Equal quantities of total cell lysates were probed with anti-1 antibody (bottom). (G) CD98 does not coimmunoprecipitate with 6 integrin. Serum-starved HMVEC-d infected with KSHV for 5 min and 10 min were immunoprecipitated with anti-6 integrin antibody and blotted for CD98hc (top). Equal quantities of total cell lysates were probed with anti-6 integrin antibody (bottom). (H) 1 integrin does not coimmunoprecipitate with CD71. Serum-starved HMVEC-d were infected with KSHV for 10 min. Uninfected and infected cell lysates were immunoprecipitated using anti-CD71 antibody and blotted for 1 integrin (top). Equal quantities of total cell lysates were probed with anti-CD71 antibodies (bottom). (I) Immunofluorescence analysis of CD98 association with V5 integrin. HMVEC-d were infected with KSHV at an MOI of 10 for 1 min, 5 min, and 10 min. The cells were fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked. Infected and uninfected cells were stained with CD98 and V5 antibodies. CD98 was visualized by incubation with Alexa 594-labeled secondary antibody (red), and V5 integrin was visualized by Alexa 488-conjugated secondary antibody (green). In the merged column, yellow shows the colocalization of integrins with CD98. Areas with colocalizing proteins are indicated by arrows. The boxed areas are enlarged in the rightmost column.

Article Snippet: Rabbit anti-LAT1 polyclonal antibody was from Novus Biologicals, Littleton, CO. Anti-goat, antirabbit, and anti-mouse antibodies linked to horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, Alexa 488, Alexa 594, and Alexa 647 were purchased from KPL Inc., Gaithersburg, MD., or Molecular Probes, Eugene, OR.

Techniques: Infection, Immunoprecipitation, Western Blot, Expressing, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Labeling

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

doi: 10.1128/jvi.01146-08

Figure Lengend Snippet: FIG. 4. Colocalization of CD98/xCT with 3, 1, and 31 integrins in KSHV-infected HMVEC-d. Serum-starved HMVEC-d were infected with KSHV at an MOI of 10 at 37°C for 10 min. Uninfected and infected cells were fixed with 4% paraformaldehyde in PBS for 30 min. The cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked, and then incubated with primary antibodies (anti-1, anti-3, anti-31, anti-CD98, anti-6, anti-xCT, or anti-LAT1) for 1 h at room temperature. The staining was visualized by incubation with Alexa 594-labeled anti-goat antibody (red) for CD98, Alexa 594-labeled anti-rabbit antibody (red) for xCT and LAT1, and anti-mouse Alexa 488-conjugated secondary antibody (green) for integrins. (A and B, C and D, E and F) Colocalization of 1, 3, and 31 integrins (green) with CD98 (red) in uninfected and infected cells, respectively. (G and H) Colocalization of 31 (green) with xCT (red) in uninfected and infected cells. (I and J) Colocalization of 31 (green) with LAT1 (red) in uninfected and infected cells. (K and L) Colocalization of 6 (green) with CD98 (red) in uninfected and infected cells. In the merged panel, yellow shows the colocalization of integrins with CD98/xCT. Areas with colocalizing proteins are indicated by arrows. Magnification, 40. The boxed areas are enlarged in the rightmost column.

Article Snippet: Rabbit anti-LAT1 polyclonal antibody was from Novus Biologicals, Littleton, CO. Anti-goat, antirabbit, and anti-mouse antibodies linked to horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, Alexa 488, Alexa 594, and Alexa 647 were purchased from KPL Inc., Gaithersburg, MD., or Molecular Probes, Eugene, OR.

Techniques: Infection, Incubation, Staining, Labeling

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

doi: 10.1128/jvi.01146-08

Figure Lengend Snippet: FIG. 8. (A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 g/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concen- tration of [3H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [3H]thymidine-labeled KSHV was prein- cubated with 100 g of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radio- activity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average standard deviation (SD) of three independent experiments. (B and C) Effects of anti- CD98 and anti-xCT peptide antibodies on KSHV DNA internaliza- tion. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h.

Article Snippet: Rabbit anti-LAT1 polyclonal antibody was from Novus Biologicals, Littleton, CO. Anti-goat, antirabbit, and anti-mouse antibodies linked to horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, Alexa 488, Alexa 594, and Alexa 647 were purchased from KPL Inc., Gaithersburg, MD., or Molecular Probes, Eugene, OR.

Techniques: Binding Assay, Incubation, Labeling, Virus, Gentle, Control, Activity Assay, Inhibition, Standard Deviation, Infection

Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

doi: 10.1128/jvi.01146-08

Figure Lengend Snippet: FIG. 9. Effects of anti-CD98 and anti-xCT antibodies on KSHV gene expression and nuclear delivery. HMVEC-d were untreated or treated with 10 g/ml of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb), with anti-CD71 antibodies (A and C), or with anti-xCT peptide antibodies (B and D) at 4°C for 1 h. The cells were washed with ice-cold PBS and then infected with KSHV at an MOI of 10 DNA copies/cell at 37°C for different times. Total RNA was isolated at 2 h, 8 h, and 24 h p.i., and 250 ng of DNase-treated RNA was subjected to real-time RT-PCR with ORF73 (A and B) and ORF50 (C and D) gene-specific primers and TaqMan probes. Known concentrations of DNase-treated in vitro-transcribed ORF50 and ORF73 transcripts were used in a real-time RT-PCR to construct a standard graph, from which the relative copy numbers of viral transcripts were calculated and normalized with GAPDH. Each reaction was done in duplicate, and each bar represents the average standard deviation (SD) of three experiments. The histograms depict the percent inhibition in RNA copy numbers for KSHV ORF73 and ORF50 genes in the presence of antibodies, which were calculated by comparison to viral-gene expression in the absence of any treatment. (E) Nuclear fractions from HMVEC-d infected at 10 DNA copies/cell for 2 h were isolated, and the purity of the nuclear fraction was confirmed by lamin B Western blots. (F) The total nuclear DNA was isolated, normalized to contain 100 ng/5 l, and analyzed by real-time DNA PCR with KSHV ORF73 primers and probe. Each reaction was done in duplicate, and each bar represents the average SD of three experiments. KSHV DNA associated with infected-cell nuclei in the absence of any treatment was considered to be 100%.

Article Snippet: Rabbit anti-LAT1 polyclonal antibody was from Novus Biologicals, Littleton, CO. Anti-goat, antirabbit, and anti-mouse antibodies linked to horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, Alexa 488, Alexa 594, and Alexa 647 were purchased from KPL Inc., Gaithersburg, MD., or Molecular Probes, Eugene, OR.

Techniques: Gene Expression, Infection, Isolation, Quantitative RT-PCR, In Vitro, Construct, Standard Deviation, Inhibition, Comparison, Western Blot