Journal: Journal of Clinical Investigation

Article Title: ZFP91 disturbs metabolic fitness and antitumor activity of tumor-infiltrating T cells

doi: 10.1172/jci144318

Figure Lengend Snippet: Figure 7. ZFP91 is required for PP2A activity in T cells. (A) Flow cytometric analysis of p-S6 levels in Zfp91+/+ Cd4-Cre (WT) and Zfp91fl/fl Cd4-Cre (KO) CD90.2+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours in the presence of SMase (0.5 U/mL) or control (50% glycerol in PBS). (B) ECAR of WT and KO T cells stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours in the presence of SMase (n = 3) or control. (C and D) Flow cytometric analysis of cell size (C) and Ki-67 expression (D, n = 3) in WT and KO T cells stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours (for FSC-A detection) or 48 hours (for Ki-67 detection) in the presence of SMase or control. (E) Flow cytometric analysis of p-S6 levels in WT and KO T cells stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours in the presence of LB-100 (1 µM) or control. (F) ECAR of WT and KO T cells stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours in the presence of LB-100 (1 µM) or control. (G and H) Flow cytometric analysis of cell size (G) and Ki-67 expression (H, n = 3) in WT and KO T cells stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours (for FSC-A detection) or 48 hours (for Ki-67 detection) in the presence of LB-100 or control. (I) IB analysis of HA-PP2Ac in WT and KO T cells transduced with either an empty vector (EV) or PP2Ac. (J–M) p-S6 expression (J), ECAR (K, n = 4), cell size (L), and Ki-67 expression (M, n = 3) for transduced T cells from I stimulated for 2 hours (J), 24 hours (K and L), or 48 hours (M) with antibodies against CD3 and CD28. Experiments were independently repeated 3 times. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-tailed Student’s t test.

Article Snippet: Antibodies against p-S6 (4858s; 1:1000 for WB), PP2Aa (2041s; 1:1000 for Western blotting [WB]), PP2Ac (2038s; 1:1000 for WB), p-Akt (Ser473) (4060s; 1:1000 for WB), p-4EBP1 (Ser65) (9451s; 1:1000 for WB), p-p70 S6 kinase (Thr389) (9234s; 1:1000 for WB), and K63 polyubiquitin (D7A11; 1:1000 for WB) were purchased from Cell Signaling Technology.

Techniques: Activity Assay, Control, Expressing, Transduction, Plasmid Preparation

Journal: Journal of Clinical Investigation

Article Title: ZFP91 disturbs metabolic fitness and antitumor activity of tumor-infiltrating T cells

doi: 10.1172/jci144318

Figure Lengend Snippet: Figure 8. ZFP91 enforces PP2A holoenzyme assembly. (A) IB analysis of ubiquitinated PP2Ac in HEK293T cells transfected with the indicated vectors. (B) IB analysis of PP2Ac K63-linked ubiquitination in Zfp91+/+ Cd4-Cre (WT) and Zfp91fl/fl Cd4-Cre (KO) CD90.2+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 4 hours. (C) Lysates from HEK293T cells transfected with the indicated vectors were subjected to IP. (D) Lysates from WT and KO CD90.2+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 4 hours were subjected to IP. (E) Lysates from WT CD90.2+ T cells stimulated with anti-CD3 and anti-CD28 antibodies were subjected to IP. (F and G) IB analysis of the indicated proteins in whole-cell lysates (WL), cytoplasmic fractions (CF), and nuclear fractions (NF) of WT CD90.2+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. (H) IB and IP assays using the cytoplasmic fractions of WT T cells stimulated with anti-CD3 and anti-CD28 antibodies. (I) Flow cytometric analysis of ZFP91 level in T cells stimulated with anti-CD3 and anti- CD28 antibodies for 24 hours. Isotype control results are shown in gray shading. US, unstimulated; ST, stimulated. (J) Flow cytometric analysis of ZFP91 expression in OT-I cells in tumors of WT mice given an i.v. injection of 2 × 106 OT-I cells on day 7 after s.c. injection of MC38 cancer cells and MC38-OVA cancer cells (n = 5). (K and L) Tumor growth (K) and survival curves (L) for Zfp91+/+ Cd4-Cre and Zfp91fl/fl Cd4-Cre mice injected s.c. with MC38 cancer cells (n = 7), followed by i.p. injection of anti–PD-1 antibody on days 7, 10, and 13. Ctrl, control antibodies. Data are representative of 3 independent experiments and are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-tailed Student’s t test (J and K) and log-rank (Mantel-Cox) test (L).

Article Snippet: Antibodies against p-S6 (4858s; 1:1000 for WB), PP2Aa (2041s; 1:1000 for Western blotting [WB]), PP2Ac (2038s; 1:1000 for WB), p-Akt (Ser473) (4060s; 1:1000 for WB), p-4EBP1 (Ser65) (9451s; 1:1000 for WB), p-p70 S6 kinase (Thr389) (9234s; 1:1000 for WB), and K63 polyubiquitin (D7A11; 1:1000 for WB) were purchased from Cell Signaling Technology.

Techniques: Transfection, Ubiquitin Proteomics, Control, Expressing, Injection

Journal: Biomolecules

Article Title: Carboxy-Methylation of the Catalytic Subunit of Protein Phosphatase 2A (PP2Ac) Integrates Methionine Availability with Methionine Addicted Cancer Cell Proliferation

doi: 10.3390/biom15091210

Figure Lengend Snippet: Methyl proteome is largely unchanged, while PP2Ac methylation is specifically sensitive to growth in −Met/+Hcy media. ( A ) MB468 and R8 cells were grown in either +Met or −Met/+Hcy media for 24 h and SAM and SAH levels were measured and compared to standard curves for absolute quantitation. The SAM/SAH ratio was calculated and indicates the methylation potential in the cells. Both met-dependent and met-independent cell lines show a significant decrease in both SAM levels and in methylation potential in response to growth in −Met/+Hcy media. * p -value less than 0.05. ( B ) Global protein methylation profiling was done in MB468 cells using liquid chromatography-mass spectrometry. No significant change in the methylproteome was observed after 3 h growth in −Met/+Hcy media. ( C ) Western blot was used to probe for methylated PP2Ac (mePP2Ac), demethylated PP2Ac (dPP2Ac), and total PP2Ac (tPP2Ac). Cells were cultured in −Met/+Hcy media for up to 24 h and harvested at different time points. PP2Ac methylation is lost as early as 2 h of media shift. ( D ) Quantification of E, using n = 3 independent experiments. ( E ) MB468 and R8 cells were grown in −Met/+Hcy media for up to 3 days, with samples being collected at different time points. Whole cell lysates were then analyzed for PP2Ac methylation. Original figures can be found in .

Article Snippet: Proteins were detected using the following primary antibodies: methyl PP2Ac (Santa Cruz, CA, USA, sc-81603, demethylated PP2Ac (Santa Cruz, CA, USA, sc-13601), total PP2Ac (Proteintech, Rosemont, IL, USA, 13482-1-AP), PARP (Cell Signaling, Danvers, MA, USA, 9542S) Cleaved Caspase-3 (Cell Signaling, 9661S), pS6 (Cell Signaling, 2317S), S6 (Cell Signaling, 4838S), LC3 A/B (Cell Signaling, 4108S), PME-1 (Millipore, MABC1183), p4EBP1 (Cell Signaling, 9459S), 4EBP1 (Cell Signaling, 9452).

Techniques: Methylation, Quantitation Assay, Liquid Chromatography, Mass Spectrometry, Western Blot, Cell Culture

Journal: Biomolecules

Article Title: Carboxy-Methylation of the Catalytic Subunit of Protein Phosphatase 2A (PP2Ac) Integrates Methionine Availability with Methionine Addicted Cancer Cell Proliferation

doi: 10.3390/biom15091210

Figure Lengend Snippet: Correlation of cell proliferation in −Met/+Hcy media and PP2Ac methylation across additional cell lines. ( A ) Methionine-independent pancreatic cancer cell line PANC1, ( B ) Methionine-independent breast cancer cell line MDA-MB-231, ( C ) Methionine-dependent pancreatic cancer cell line BxPC3. All cells were all cultured in −Met/+Hcy media and PP2A methylation was analyzed after 3 and 24 h of treatment. Cell proliferation was also measured for 6 days in either +Met or −Met+Hcy media. Original figures can be found in .

Article Snippet: Proteins were detected using the following primary antibodies: methyl PP2Ac (Santa Cruz, CA, USA, sc-81603, demethylated PP2Ac (Santa Cruz, CA, USA, sc-13601), total PP2Ac (Proteintech, Rosemont, IL, USA, 13482-1-AP), PARP (Cell Signaling, Danvers, MA, USA, 9542S) Cleaved Caspase-3 (Cell Signaling, 9661S), pS6 (Cell Signaling, 2317S), S6 (Cell Signaling, 4838S), LC3 A/B (Cell Signaling, 4108S), PME-1 (Millipore, MABC1183), p4EBP1 (Cell Signaling, 9459S), 4EBP1 (Cell Signaling, 9452).

Techniques: Methylation, Cell Culture

Journal: Biomolecules

Article Title: Carboxy-Methylation of the Catalytic Subunit of Protein Phosphatase 2A (PP2Ac) Integrates Methionine Availability with Methionine Addicted Cancer Cell Proliferation

doi: 10.3390/biom15091210

Figure Lengend Snippet: The growth defect of methionine-dependent MB468 cells in −Met/+Hcy media is not mediated by mTor signaling. ( A ) MB468 cells were cultured with either methionine depletion (no homocysteine supplementation), leucine depletion, or 100 nM Rapamycin for 24 h. Whole cell lysates were analyzed for both PP2Ac methylation and S6 phosphorylation. ( B ) MB468 and R8 cells were grown in −Met/+Hcy media for up to 3 days, with samples being collected at different time points. Whole cell lysates were tested for mTORC1 activity by analyzing phosphorylation of mTOR targets. ( C ) LC3II/I ratios indicate that growth in −Met/+Hcy media does not induce autophagy. * p -value less than 0.05. Original figures can be found in .

Article Snippet: Proteins were detected using the following primary antibodies: methyl PP2Ac (Santa Cruz, CA, USA, sc-81603, demethylated PP2Ac (Santa Cruz, CA, USA, sc-13601), total PP2Ac (Proteintech, Rosemont, IL, USA, 13482-1-AP), PARP (Cell Signaling, Danvers, MA, USA, 9542S) Cleaved Caspase-3 (Cell Signaling, 9661S), pS6 (Cell Signaling, 2317S), S6 (Cell Signaling, 4838S), LC3 A/B (Cell Signaling, 4108S), PME-1 (Millipore, MABC1183), p4EBP1 (Cell Signaling, 9459S), 4EBP1 (Cell Signaling, 9452).

Techniques: Cell Culture, Methylation, Phospho-proteomics, Activity Assay

Journal: Biomolecules

Article Title: Carboxy-Methylation of the Catalytic Subunit of Protein Phosphatase 2A (PP2Ac) Integrates Methionine Availability with Methionine Addicted Cancer Cell Proliferation

doi: 10.3390/biom15091210

Figure Lengend Snippet: Reducing PP2Ac methylation is sufficient to impair cell proliferation in −Met/+Hcy media. ( A , B ) The protein phosphatase methylesterase (PME-1) was overexpressed to decrease PP2Ac methylation in cells. Cell proliferation in +Met or −Met/+Hcy media was compared in both parental and PME-1 overexpressing (PME-1 OE) cells. ( A ) Reducing PP2Ac methylation in R8 cells induced proliferation defects in met-independent R8 cells, while met-dependent MB468 cells were unaffected by PME-1 overexpression. ( B ) Met-independent R1 cells were severely impaired in proliferation in −Met/+Hcy media when PP2Ac methylation was reduced, but HEK293T cells were unaffected by PME-1 overexpression. Note: HA-PP2A- one allele of PP2A was HA-tagged on the endogenous locus. PP2A- endogenous PP2A ( C ) Methionine-independent R8 cells were transduced with myc-PP2Ac ∆Leu309 to mimic demethylation. Growth rates between parental R8, the pool of ∆Leu309 cells, and two individual ∆Leu309 colonies were compared. An increase in demethylated PP2Ac is enough to impair proliferation in −Met/+Hcy media. ∆Leu Col2 had very low expression of the PP2Ac ∆Leu309 allele that was visible only after very long exposure. ( D ) Expression of myc-PP2Ac ∆Leu309 in methionine-independent R1 cells is sufficient to impair proliferation in −Met/+Hcy media. Original figures can be found in .

Article Snippet: Proteins were detected using the following primary antibodies: methyl PP2Ac (Santa Cruz, CA, USA, sc-81603, demethylated PP2Ac (Santa Cruz, CA, USA, sc-13601), total PP2Ac (Proteintech, Rosemont, IL, USA, 13482-1-AP), PARP (Cell Signaling, Danvers, MA, USA, 9542S) Cleaved Caspase-3 (Cell Signaling, 9661S), pS6 (Cell Signaling, 2317S), S6 (Cell Signaling, 4838S), LC3 A/B (Cell Signaling, 4108S), PME-1 (Millipore, MABC1183), p4EBP1 (Cell Signaling, 9459S), 4EBP1 (Cell Signaling, 9452).

Techniques: Methylation, Over Expression, Transduction, Expressing

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 3. FTY720 binds SET similarly as D-e-C18 ceramide. A) Overlaid [1H-1H] total correlational spectroscopy spectra of FTY720. Quantitation of line broadening as a function of peak volume and increasing concentration of Nd-SETWT (0–50 mM). Chemical structure of functional groups on FTY720 affected by the addition of Nd-SET. B) CSP of NdCd-SET induced by 3 mM (black bars), 7 mM (red bars), and 10 mM FTY720 (blue bars) as a function of assigned residue. CSP titrations were performed in triplicate. Red line is calculated as 3s, and any value .3s is considered a significant shift. C) Model of FTY720 bound SET. Residues colored red underwent a significant chemical shift during NMR titration analysis. D) Association between endogenous SET, FLAG-SETWT, FLAG-SETE111A, and FLAG-SETR71A mutants and PP2AC 6 5 mM FTY720 was monitored by PLA using antibodies against SET, FLAG, or PP2AC. SET mutants were generated from CSP and modeling. Representative images of n $ 3 independent experiments per group. Scale bar, 100 mm. E) Quantitation of PLA for SET mutants designed to limit response to FTY720 treatment. Data are means 6 SEM of n $3 independent experiments per group, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, ***P , 0.001.

Article Snippet: Protein extracts were analyzed by SDS-PAGE, transferred to PVDFmembrane, andprobedwith the followingantibodies: SET (Bethyl Laboratories, Montgomery, TX, USA), PP2AC (1D6; MilliporeSigma), PP2AC (C1, sc376673), PP2AC (2259S, 52F8D8; Cell Signaling Technology, Danvers, MA, USA), FLAG (F7425; MilliporeSigma), B56g (sc375380; Santa Cruz Biotechnology, Dallas, TX, USA), B56g (sc81605; Santa Cruz Biotechnology), HA-tag (C29F4; Cell Signaling Technology), b-Actin (2066; MilliporeSigma), p-PP2AC Tyr307 (sc271903; Santa Cruz Biotechnology), and V5 epitope (V8012; MilliporeSigma).

Techniques: Spectroscopy, Quantitation Assay, Concentration Assay, Functional Assay, Residue, Titration, Generated

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 5. SET interacts with specific PP2A holoenzyme. A–C) Association between endogenous SET and PP2AC in shPP2AA⍺and shPP2AAb (A) knockdown cells, interaction between endogenous SET and PP2AC in shB56g and shB56d (B) knockdown cells, and association between endogenous SET and PP2AC in shPP2AC⍺and shPP2ACb (C) knockdown cells was assessed by PLA. Data are means 6 SEM of n 5 3 independent experiments, analyzed by Student’s t test. *P , 0.05, **P , 0.01. D) Western blot showing successful reconstitution of expression for V5-PP2AA⍺and V5-PP2AAb. E) Interaction between endogenous SET and PP2AC in shPP2AA⍺and shPP2AAb stable knockdown cells with rescued expression (V5-PP2AA⍺and V5-PP2AAb was assessed by (continued on next page)

Article Snippet: Protein extracts were analyzed by SDS-PAGE, transferred to PVDFmembrane, andprobedwith the followingantibodies: SET (Bethyl Laboratories, Montgomery, TX, USA), PP2AC (1D6; MilliporeSigma), PP2AC (C1, sc376673), PP2AC (2259S, 52F8D8; Cell Signaling Technology, Danvers, MA, USA), FLAG (F7425; MilliporeSigma), B56g (sc375380; Santa Cruz Biotechnology, Dallas, TX, USA), B56g (sc81605; Santa Cruz Biotechnology), HA-tag (C29F4; Cell Signaling Technology), b-Actin (2066; MilliporeSigma), p-PP2AC Tyr307 (sc271903; Santa Cruz Biotechnology), and V5 epitope (V8012; MilliporeSigma).

Techniques: Knockdown, Western Blot, Expressing

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 7. Myosin IIa is a potential target of FTY720-activated PP2A. A) Validation of PP2AC/myosin IIa association observed in SILAC data by coimmunoprecipitation. Exogenous expression of HA-PP2ACaWT in A549 cells using magnetic HA-conjugated beads 6 treatment of 20 mM FTY720 for 2 h. B) Association between PP2AC and myosin IIa in shSCR and shB56g stable knockdown cell lines 6 treatment of 5 mM FTY720 for 3 h by PLA using antibodies against PP2AC and myosin IIa. Quantitation of PLA experiments. Data are means 6 SEM of n = 3 independent experiments, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, **P , 0.01. C) PP2A activity assay in shSCR and shB56g stable knockdown cell lines 6 treatment of 5 mM FTY720 for 16 h using purified rabbit MYH9 as a substrate. Data are means 6 SD of n = 3 independent experiments analyzed by 2-way ANOVA (continued on next page)

Article Snippet: Protein extracts were analyzed by SDS-PAGE, transferred to PVDFmembrane, andprobedwith the followingantibodies: SET (Bethyl Laboratories, Montgomery, TX, USA), PP2AC (1D6; MilliporeSigma), PP2AC (C1, sc376673), PP2AC (2259S, 52F8D8; Cell Signaling Technology, Danvers, MA, USA), FLAG (F7425; MilliporeSigma), B56g (sc375380; Santa Cruz Biotechnology, Dallas, TX, USA), B56g (sc81605; Santa Cruz Biotechnology), HA-tag (C29F4; Cell Signaling Technology), b-Actin (2066; MilliporeSigma), p-PP2AC Tyr307 (sc271903; Santa Cruz Biotechnology), and V5 epitope (V8012; MilliporeSigma).

Techniques: Biomarker Discovery, Multiplex sample analysis, Expressing, Knockdown, Quantitation Assay, Activity Assay

Journal: The FASEB Journal

Article Title: The NMR‐based characterization of the FTY720‐SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET‐PP2A interaction

doi: 10.1096/fj.201802264r

Figure Lengend Snippet: Figure 8. Phosphorylation of SET influences PP2A activity. A) Association between endogenous SET, FLAG-SETWT, FLAG- SETS171A, and FLAG-SETS171E, mutants, and PP2AC 6 5 mM FTY720 with a pCDH empty vector control measured by PLA using antibodies against SET, FLAG, and PP2AC. Data are means 6 SEM of n = 3 independent experiments, analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, **P , 0.01, ****P , 0.0001. B) Association between FLAG-SETS171A and FLAG-SETS171E and B56g 6 5 mM FTY720 assessed by PLA using antibodies against FLAG and B56g. Data are means 6 SEM of n = 3 independent experiments analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05, **P , 0.01. C) Western blot of PP2A-p-Tyr307 of PP2AC as a marker of activity normalized to b-actin and FLAG expression. Representative blot of n = 3 individual experiments. D) Blots were quantified with ImageJ. Data are means normalized to actin and FLAG 6 SD and analyzed by 2-way ANOVA with Tukey’s post hoc test. *P , 0.05. E) Quantification of GA cross-linking of FLAG-SETWT, FLAG-SETS171A, and FLAG-SETS171E

Article Snippet: Protein extracts were analyzed by SDS-PAGE, transferred to PVDFmembrane, andprobedwith the followingantibodies: SET (Bethyl Laboratories, Montgomery, TX, USA), PP2AC (1D6; MilliporeSigma), PP2AC (C1, sc376673), PP2AC (2259S, 52F8D8; Cell Signaling Technology, Danvers, MA, USA), FLAG (F7425; MilliporeSigma), B56g (sc375380; Santa Cruz Biotechnology, Dallas, TX, USA), B56g (sc81605; Santa Cruz Biotechnology), HA-tag (C29F4; Cell Signaling Technology), b-Actin (2066; MilliporeSigma), p-PP2AC Tyr307 (sc271903; Santa Cruz Biotechnology), and V5 epitope (V8012; MilliporeSigma).

Techniques: Phospho-proteomics, Activity Assay, Plasmid Preparation, Control, Western Blot, Marker, Expressing