Journal: Journal of cell science

Article Title: Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

doi: 10.1242/jcs.195867

Figure Lengend Snippet: Fig. 1. The effects of nuclear actin filaments on nucleoplasmic actin. (A) Non-transfected cells or COS7 cells transfected with (B) V163M-α-actin–GFP, (C) the EGFP–supervillin fragment or (D) EYFP–NLS-β-actin (all shown in green) form large phalloidin-stained (red) actin filaments throughout the nucleus (blue). Scale bars: 5 µm. (E) FRAP experiment performed on COS7 cells transfected with EYFP–NLS-β-actin for 48 h. Areas of diffuse nuclear EYFP–β-actin were bleached in transfected cells (white circle and arrow) that had [nuclear actin filament (NAF) positive] and did not have (NAF negative) nuclear actin filaments. (F) FRAP analysis of nuclear pools of EYFP–β-actin fitted to a biphasic exponential recovery curve. Results are mean±s.e.m. Nuclear-actin-filament-positive nuclei (green) exhibit substantially longer recovery kinetics than those without filaments (nuclear actin filament negative; gray). FRAP kinetics are listed in Table S1A.

Article Snippet: Primary antibodies were purchased as shown: RNAPII antibodies, H5, H14 (western blotting, 1:5000) (Covance), 4H8 (immunofluorescence, 1:150; western blotting, 1:5000; immunoprecipitation, 5 μg) (Abcam and Active Motif ); β-actin antibodies Ac-15 (immunofluorescence, 1:200; western blotting, 1:10,000; immunoprecipitation, 5 μg) and Ac-40 (immunofluorescence, 1:100; western blotting, 1:1000) (Sigma-Aldrich) and pan-actin C4 (immunofluorescence, 1:100; western blotting, 1:5000) (EMD Millipore); non-specific IgG (immunoprecipitation, 5 μg) (Santa Cruz Biotechnology); BrdU/BrU (immunofluorescence, 1:200) (BU-1, EMD Millipore), GFP (immunoprecipitation, 3 μg) (Abcam) and SC-35 (immunofluorescence, 1:100) (SRp30b, EMD Millipore).

Techniques: Transfection, Staining

Journal: Journal of cell science

Article Title: Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

doi: 10.1242/jcs.195867

Figure Lengend Snippet: Fig. 3. Nuclear actin filament formation inhibits transcription and proliferation. (A) COS7 cells transfected as shown (green) were treated with 2 mM BrU, stained for BrU incorporation (red) and normalized to non-transfected cells (asterisks). Quantification of BrU fluorescence is shown on the right. Fluorescence intensity was normalized to non-transfected cells in each frame. Results are mean±s.e.m. (n=3, >300 cells/group). **P<0.01 (one-way ANOVA). (B) COS7 cells co-transfected with the mCherry–supervillin fragment and either wild-type EYFP–R62D-β-actin or EYFP–NLS-R62D-β-actin for 48 h then treated with BrU as in A. Arrowheads denote nuclei with filaments. Quantification of the fluorescence intensity shows increasing R62D-actin levels in the nucleus, but not in the cytoplasm, is able to restore transcription levels. Fluorescence intensity was normalized to non-transfected cells in each frame (Tukey box plot indicates the 25–75th percentiles, and the median; the whiskers show 1.5 times the interquartile distance; dots represent outliers). ****P<0.0001; n.s., not significant (one-way ANOVA). (C) Transfected COS7 cells were treated with 10 µM BrdU for 5 h, stained for BrdU incorporation (red), and normalized to non-transfected cells (asterisks). Quantification of BrU fluorescence is shown on the right. Fluorescence intensity was normalized to non-transfected cells in each frame. Results are mean± s.e.m. (n=5, >400 cells/group). ***P<0.001 (one-way ANOVA).

Article Snippet: Primary antibodies were purchased as shown: RNAPII antibodies, H5, H14 (western blotting, 1:5000) (Covance), 4H8 (immunofluorescence, 1:150; western blotting, 1:5000; immunoprecipitation, 5 μg) (Abcam and Active Motif ); β-actin antibodies Ac-15 (immunofluorescence, 1:200; western blotting, 1:10,000; immunoprecipitation, 5 μg) and Ac-40 (immunofluorescence, 1:100; western blotting, 1:1000) (Sigma-Aldrich) and pan-actin C4 (immunofluorescence, 1:100; western blotting, 1:5000) (EMD Millipore); non-specific IgG (immunoprecipitation, 5 μg) (Santa Cruz Biotechnology); BrdU/BrU (immunofluorescence, 1:200) (BU-1, EMD Millipore), GFP (immunoprecipitation, 3 μg) (Abcam) and SC-35 (immunofluorescence, 1:100) (SRp30b, EMD Millipore).

Techniques: Transfection, Staining, Fluorescence, BrdU Incorporation Assay

Journal: Journal of cell science

Article Title: Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

doi: 10.1242/jcs.195867

Figure Lengend Snippet: Fig. 4. Nuclear actin filament formation impairs RNAPII localization and dynamics. Structured illumination microscopy of (A) control and cells transfected with (B) the EGFP–supervillin fragment, (C) EYFP–NLS-β-actin, (D) V163M-α-actin–GFP, and stained with anti-phospho-RNAPII (4H8) antibody. White arrowheads show large RNAPII clusters in transfected cells. Scale bars: 5 µm. (E) RNAPII foci area (# square pixels) in non-transfected, mCherry–supervillin- fragment-transfected, and cells co-transfected with the mCherry–supervillin fragment and EYFP–NLS-R62D-β-actin was graphed to log scale using a box plot (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the min. and max. values); >2500 foci were mapped in each group. Demonstrative images are shown in Fig. S3A. (F) FRAP analyses of RNAPII dynamics of COS7 cells stably expressing GFP-RNAPII. FRAP curves show data points as mean± s.e.m. and recovery lines of best fit. FRAP kinetics are listed in Table S1B. FRAP analyses were performed on small RNAPII transcription factories in control cells (green data points, dotted line) or on cells co-transfected with the mCherry–supervillin fragment that exhibit small transcription factories (blue data points, dashed line) and large factories (red data points, solid line). Large factories exhibited a slower fluorescence recovery. In each micrograph, small foci are marked with blue circles, whereas medium (control nuclei) and large foci (nuclear-actin-filament-positive nuclei) are marked with white circles. Scale bars: 5 µm. (G) U2OS-263 cells co-transfected with MS2–GFP and the mCherry–supervillin fragment. FRAP analysis (mean±s.e.m.) of cells transfected with the mCherry–supervillin fragment (red and gray) show decreased MS2–GFP fluorescence recovery as compared to control cells (blue and black). FRAP kinetics are listed in Table S1C.

Article Snippet: Primary antibodies were purchased as shown: RNAPII antibodies, H5, H14 (western blotting, 1:5000) (Covance), 4H8 (immunofluorescence, 1:150; western blotting, 1:5000; immunoprecipitation, 5 μg) (Abcam and Active Motif ); β-actin antibodies Ac-15 (immunofluorescence, 1:200; western blotting, 1:10,000; immunoprecipitation, 5 μg) and Ac-40 (immunofluorescence, 1:100; western blotting, 1:1000) (Sigma-Aldrich) and pan-actin C4 (immunofluorescence, 1:100; western blotting, 1:5000) (EMD Millipore); non-specific IgG (immunoprecipitation, 5 μg) (Santa Cruz Biotechnology); BrdU/BrU (immunofluorescence, 1:200) (BU-1, EMD Millipore), GFP (immunoprecipitation, 3 μg) (Abcam) and SC-35 (immunofluorescence, 1:100) (SRp30b, EMD Millipore).

Techniques: Microscopy, Control, Transfection, Staining, Stable Transfection, Expressing, Fluorescence

Journal: Journal of cell science

Article Title: Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

doi: 10.1242/jcs.195867

Figure Lengend Snippet: Fig. 5. Nuclear actin filament formation reduces the interaction of actin with RNAPII and RNAPII gene recruitment. (A) GFP immunoprecipitation (IP) assay on HeLa cells transfected with EGFP or EYFP–NLS-β-actin with the S14C (polymerization promoting) or R62D (polymerization resistant) mutation. WB, western blotting. Quantification of the ratio of RNAPII levels to GFP levels shows that RNAPII (H14 antibody) associates better with the polymerization-resistant (R62D) mutant. Data were normalized to the ratio of RNAPII and GFP in the input lanes. Results are mean±s.e.m. (n=4). **P<0.01 (one-way ANOVA). (B) Co- immunoprecipitation assay of HeLa cells transfected with GFP or constructs that led to the formation of nuclear actin filaments and immunoprecipitated with antibodies to endogenous β-actin (left) or RNAPII (4H8 antibody, right) and blotted for RNAPII (H14 antibody) and β-actin. The quantification of β-actin to RNAPII levels shows a reduced interaction between endogenous β-actin and RNAPII in cells containing nuclear actin filament as compared to cells with EGFP alone. Data were normalized to the ratio of RNAPII and GFP in the input lanes. Results are mean±s.e.m. (n=4). *P<0.05; **P<0.01 (one-way ANOVA). (C) ChIP-qPCR analysis of the recruitment of RNAPII to the activated MHCIITA promoter in cells containing nuclear actin filaments. Data were calculated as a percentage of input and normalized to enrichment in non-induced cells. Results are mean±s.e.m. (n=4). *P<0.05; **P<0.01 (one-way ANOVA). (D) Luciferase assay of the MHCIITA promoter as a measure of gene activation in cells expressing actin controls or in cells with nuclear actin filaments. Relative luciferase units per µg of protein in each lysate was calculated and normalized to cells transfected with only the MHCIITA promoter luciferase construct. Results are mean± s.e.m. (n=6). *P<0.05; **P<0.01; ***P<0.001; n.s., not significant (t-test).

Article Snippet: Primary antibodies were purchased as shown: RNAPII antibodies, H5, H14 (western blotting, 1:5000) (Covance), 4H8 (immunofluorescence, 1:150; western blotting, 1:5000; immunoprecipitation, 5 μg) (Abcam and Active Motif ); β-actin antibodies Ac-15 (immunofluorescence, 1:200; western blotting, 1:10,000; immunoprecipitation, 5 μg) and Ac-40 (immunofluorescence, 1:100; western blotting, 1:1000) (Sigma-Aldrich) and pan-actin C4 (immunofluorescence, 1:100; western blotting, 1:5000) (EMD Millipore); non-specific IgG (immunoprecipitation, 5 μg) (Santa Cruz Biotechnology); BrdU/BrU (immunofluorescence, 1:200) (BU-1, EMD Millipore), GFP (immunoprecipitation, 3 μg) (Abcam) and SC-35 (immunofluorescence, 1:100) (SRp30b, EMD Millipore).

Techniques: Immunoprecipitation, Transfection, Mutagenesis, Western Blot, Co-Immunoprecipitation Assay, Construct, ChIP-qPCR, Luciferase, Activation Assay, Expressing

Journal: Journal of cell science

Article Title: Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

doi: 10.1242/jcs.195867

Figure Lengend Snippet: Fig. 6. Sequestering nuclear actin inhibits transcription in vitro. (A) Representative immunoblot of HeLa nuclear extract pre-incubated with buffer or 0.5 µM ACD to crosslink nuclear actin, and probed with antibodies to β-actin (top) and RNAPII (bottom). WB, western blotting. (B) Graph of 32[P]-labeled RNA transcripts from an adenovirus major late promoter cassette incubated with HeLa nuclear extract pre-treated with buffer or 0.5 µM ACD. A representative autoradiograph is shown. Quantification of transcript band density normalized to HeLa nuclear extract alone shows a significant decrease in transcription in ACD-treated extracts. Results are mean±s.e.m. (n=5). *P<0.05 (t-test). (C) Immunoprecipitation (IP) of RNAPII (4H8 antibody) from HeLa cell nuclear extract treated with buffer or ACD and probed with β-actin (top) and RNAPII (H14) antibody (bottom). Note the lack of high-molecular-mass actin species in the ACD-treated immunoprecipitate and lower levels of associated monomeric β-actin. (D) 100,000 g sedimentation assays of purified HeLa nuclear extract pre-treated with DMSO or 10 µM phalloidin. (E) Graph of 32[P]-labeled RNA transcripts as in B pre-treated with DMSO, 2 µM, or 10 µM phalloidin. A representative autoradiograph is shown. Quantification of transcript band density, normalized to untreated HeLa nuclear extract, shows significant decreases in transcription in extracts treated with 10 µM phalloidin. Results are mean±s.e.m. (n=3). *P<0.05 (one-way ANOVA). (F) Immunoprecipitation of RNAPII (4H8 antibody) from HeLa nuclear extract after treatment with vehicle or 10 µM phalloidin. Western blots were probed with RNAPII (H14) antibody (top) and β-actin (bottom).

Article Snippet: Primary antibodies were purchased as shown: RNAPII antibodies, H5, H14 (western blotting, 1:5000) (Covance), 4H8 (immunofluorescence, 1:150; western blotting, 1:5000; immunoprecipitation, 5 μg) (Abcam and Active Motif ); β-actin antibodies Ac-15 (immunofluorescence, 1:200; western blotting, 1:10,000; immunoprecipitation, 5 μg) and Ac-40 (immunofluorescence, 1:100; western blotting, 1:1000) (Sigma-Aldrich) and pan-actin C4 (immunofluorescence, 1:100; western blotting, 1:5000) (EMD Millipore); non-specific IgG (immunoprecipitation, 5 μg) (Santa Cruz Biotechnology); BrdU/BrU (immunofluorescence, 1:200) (BU-1, EMD Millipore), GFP (immunoprecipitation, 3 μg) (Abcam) and SC-35 (immunofluorescence, 1:100) (SRp30b, EMD Millipore).

Techniques: In Vitro, Western Blot, Incubation, Labeling, Autoradiography, Immunoprecipitation, Sedimentation, Purification

Journal: bioRxiv

Article Title: Nuclear PHGDH promotes neutrophil recruitment to drive liver cancer progression

doi: 10.1101/2021.10.17.464745

Figure Lengend Snippet: (A) Volcano plot of the proteins differentially interacting with Phgdh as determined by the ratio between the MET/CAT-induced liver cancer group and the mock group in W2 and W5. The mice in the mock group were injected with blank control plasmids. cMyc is shown as brown red circles. (B) Co-immunoprecipitation (Co-IP) assays were performed with an antibody against Phgdh followed by Western blot analysis of Phgdh to detect the interaction between Phgdh and cMyc. Loading control, β–actin. (C) C57BL/6 mice were injected with NCT-503 (dosage, 100 mg/kg) through the tail vein in W2 and W5, and 12 hours later after injection, mouse liver tissues were dissected for Co-IP assay to detect the interaction between Phgdh and cMyc. Loading control, β–actin. (D) Separation of nuclei and cytosol was performed using PLC/PRF/5 and Hep3B cells. Co-IP assay was conducted using nuclei to detect the interaction between Phgdh and cMyc. Cyto, cytosol; Nuc, nuclei. (E) The construction of nuclear export sequence (NES) tagged PHGDH (PHGDH-NES) in its C terminal was validated by immunoblotting in PLC/PRF/5 and Hep3B cells. shNT, a non-targeting short hairpin RNA (shRNA); shPHGDH, shRNA against PHGDH; rPHGDH-WT, shRNA-resistant wild type PHGDH; rPHGDH-NES, shRNA-resistant PHGDH-NES. Loading control, β–actin. (F) Separation of nuclei and cytosol was performed using PHGDH-depleted PLC/PRF/5 and Hep3B cells rescued with rPHGDH-WT or rPHGDH-NES. Co-IP assay was conducted using nuclei to detect the interaction between Phgdh and cMyc. (G) Immunofluorescence assay using PHGDH-depleted Hep3B cells rescued with rPHGDH-WT or rPHGDH-NES. Cells were fixed with 80% methanol and stained with antibodies against cMyc or PHGDH. 4′, 6-Diamidino-2-phenylindole (DAPI) was used as a nuclear localization marker (left panel). Scale bars: 20 μm. The fluorescence intensity profile of regions of interest (the dotted lines) was quantified to illustrate the colocalization of PHGDH and cMyc using ImageJ (right panel). (H) cMyc transactivation was measured with a Dual-Luciferase ® Reporter Assay System according to the manual using cells from E . (Mean ± SD, one-way ANOVA followed by Dunnett’s multiple comparisons test, n = 3)

Article Snippet: Mouse monoclonal antibodies against cMyc (sc-40, 1:2,000 for IB, 1:200 for IHC and 1:50 for ChIP), tubulin (sc-5286, 1:2,000 for IB) and CYP2B10 (sc-73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

Techniques: Injection, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Sequencing, shRNA, Immunofluorescence, Staining, Marker, Fluorescence, Luciferase, Reporter Assay

Journal: bioRxiv

Article Title: Nuclear PHGDH promotes neutrophil recruitment to drive liver cancer progression

doi: 10.1101/2021.10.17.464745

Figure Lengend Snippet: (A) Schematic diagram of human PHGDH protein and its five domains. aa, amino acid. (B) HA-tagged cMyc and FLAG-tagged PHGDH (including WT and five domain truncates) were transiently transfected into HEK293T cells. Co-IP was performed with an antibody against FLAG. Antibodies against HA and FLAG were used to detect the association between cMyc and PHGDH. HA was used as an imput control. dSB1, SB1 domain depletion; dNB, NB domain depletion; dSB2, SB2 domain depletion; dASB, ASB domain depletion; dACT, ACT domain depletion. (C) GST-tagged ACT domain and His-tagged cMyc protein were purified from E. coli , and a GST pull-down assay was performed by incubating both the recombinant proteins together. GST was used as a blank control. Antibodies against His and GST were used to detect the association between cMyc and the ACT domain. (D) cMyc transcriptional activity was measured by the Dual-Luciferase ® Reporter Assay System according to the manual using PHGDH-depleted PLC/PRF/5 and Hep3B cells rescued with rPHGDH-WT or rPHGDH-dACT. Cells expressing shNT were used as control. rPHGDH-dACT, shRNA-resistant PHGDH-dACT. (Mean ± SD, one-way ANOVA followed by Dunnett’s multiple comparisons test, n = 3) (E) Co-IP analysis of p300, cMyc and PHGDH was performed using PHGDH-depleted PLC/PRF/5 cells rescued with rPHGDH-WT or rPHGDH-dACT. Antibody against p300 was used to enrich p300 associated complex. Immunoblotting analysis of PHGDH, cMyc and p300 was performed using the indicated antibodies. (F) p300 was transiently depleted by specific siRNA in PHGDH-depleted PLC/PRF/5 cells rescued with rPHGDH-WT or rPHGDH-dACT. Immunoblotting analysis of cMyc-AcK148, cMyc and p300 was performed using the indicated antibodies. NC, negative control. (G) Co-IP analysis of cMyc, p300 and PHGDH was performed using PHGDH-depleted PLC/PRF/5 cells rescued with rPHGDH-WT or rPHGDH-dACT. Antibody against cMyc was used to enrich cMyc associated complex. Immunoblotting analysis of PHGDH, p300, cMyc and AF9 was performed using the indicated antibodies.

Article Snippet: Mouse monoclonal antibodies against cMyc (sc-40, 1:2,000 for IB, 1:200 for IHC and 1:50 for ChIP), tubulin (sc-5286, 1:2,000 for IB) and CYP2B10 (sc-73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

Techniques: Transfection, Co-Immunoprecipitation Assay, Control, Purification, Pull Down Assay, Recombinant, Activity Assay, Luciferase, Reporter Assay, Expressing, shRNA, Western Blot, Negative Control

Journal: bioRxiv

Article Title: Nuclear PHGDH promotes neutrophil recruitment to drive liver cancer progression

doi: 10.1101/2021.10.17.464745

Figure Lengend Snippet: (A-D) RNA-sequencing analyses were performed using PHGDH-depleted PLC/PRF/5 cells rescued with rPHGDH-WT or rPHGDH-dACT. Gene Ontology (GO) enrichment analyses of the differentially expressed genes were presented (A) . GSEA enrichment plot of the KEGG pathway NOD-like receptor-related gene was shown in (B) . The correlation of all NOD-like receptor-related gene expression with the phgdh expression status was displayed by the ranking metric score. A positive score indicates a correlation with the rPHGDH-dACT and a negative score indicates a correlation with rPHGDH-WT; The red indicates a gene that contributes most to the enrichment result and the blue indicates a gene that contributes less (C) . The total differentially expressed genes (FC>2 or FC<0.5; p value<0.05) were displayed using a volcano plot. FC, fold change of rPHGDH-dACT compared to rPHGDH-WT (D). (E) qRT-PCR validated the top up-regulated genes from C . (Mean ± SD, two-tailed Student’s t-test, n = 3). (F) ELISA examined the concentration of CXCL1/8 and IL1B in the medim culturing PHGDH-depleted PLC/PRF/5 cells, which were rescued with rPHGDH-WT or rPHGDH-dACT. (Mean ± SD, two-tailed Student’s t-test, n = 3) (G) Immunoblotting analysis of CXCL1/8, PHGDH and cMyc was performed using the indicated cells and antibodies. (H) Co-IP analysis of cMyc, p300 and PHGDH was performed using PLC/PRF/5 cells expressing WT or K148R mutant Myc. Antibody against cMyc was used to enrich cMyc associated complex. Immunoblotting analysis of PHGDH, p300, cMyc and AF9 was performed using the indicated antibodies. (I) qRT-PCR validated CXCL1/8 and IL1B genes using PLC/PRF/5 cells expressing WT or K148R mutant Myc. (Mean ± SD, two-tailed Student’s t-test, n = 3). (J) ELISA examined the concentration of CXCL1/8 and IL1B in the medim culturing PLC/PRF/5 cells expressing WT or K148R mutant Myc. (Mean ± SD, two-tailed Student’s t-test, n = 3). (K) Immunoblotting analysis of CXCL1/8, PHGDH and cMyc was performed using the indicated cells and antibodies. (L) ChIP analysis of PHGDH, cMyc, p300, RNA Pol II, AF9 and H3Kac on CXCL1 gene promoter was performed using indicated cells. IgG was used as a blank control. (Mean ± SD, two-tailed Student’s t-test, n = 3)

Article Snippet: Mouse monoclonal antibodies against cMyc (sc-40, 1:2,000 for IB, 1:200 for IHC and 1:50 for ChIP), tubulin (sc-5286, 1:2,000 for IB) and CYP2B10 (sc-73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

Techniques: RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Control

Journal: bioRxiv

Article Title: Nuclear PHGDH promotes neutrophil recruitment to drive liver cancer progression

doi: 10.1101/2021.10.17.464745

Figure Lengend Snippet: (A) Comparation of PHGDH expression levels in HCC tumors and normal adjacent tissues (NATs) using the TCGA dataset. (Mann-Whitney test) (B) Comparation of PHGDH protein levels in HCC tumors and paired normal adjacent tissues (NATs) using the two HCC proteomic datasets (Gao et al. 2019, Cell ; Jiang et al. 2019, Nature ). (Wilcoxon matched-pairs signed rank test) (C) IHC staining of PHGDH in paired human liver tissues (NAT) and liver cancer tissues (n = 25, Wilcoxon matched-pairs signed rank test). Representative images of PHGDH staining in paired samples were shown. Scale bars: 50 μm. (D) IHC staining of PHGDH in human liver cancer tissues. Representative images of nucleus (Nuc), cytoplasma (Cyto), both nucleus and cytoplasma (Nuc & Cyto) localized PHGDH were shown. Scale bars: 50 μm (left); 10 μm (right). (E) The patient counts of different subcellular localized PHGDH in human liver cancer tissues (upper). Kaplan-Meier plot showing the survival of patients with different subcellular signals of PHGDH. (Log-rank test) (lower). (F) The positive correlation of PHGDH and cMyc, or PHGDH and cMyc-AcK148 in clinical liver cancer patients were examined by IHC. Right panels show the semi-quantitative scoring (using a scale from 0 to 5) between two staining signals was carried out (Pearson product moment correlation test). Scale bar, 50 μm (G) Survival plot of PHGDH expression, cMyc expression, the combined expression of PHGDH and cMyc in TCGA-LIHC data with median cutoff. (Log-rank test.) (H) Work model of the nonmetabolic role of PHGDH.

Article Snippet: Mouse monoclonal antibodies against cMyc (sc-40, 1:2,000 for IB, 1:200 for IHC and 1:50 for ChIP), tubulin (sc-5286, 1:2,000 for IB) and CYP2B10 (sc-73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

Techniques: Expressing, MANN-WHITNEY, Immunohistochemistry, Staining