mouse monoclonal antibody against c myc  (Proteintech)

Journal: Advanced Science

Article Title: Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer

doi: 10.1002/advs.202205818

Figure Lengend Snippet: Phgdh interacts with cMyc independent of its enzyme activity. 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. Transcriptional factors, including cMyc, were 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) 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. D) 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. E) 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. F) 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). G) cMyc transactivation was measured with a Dual‐Luciferase Reporter Assay System according to the manual using cells from (D) (mean ± SD, one‐way ANOVA followed by Dunnett's multiple comparisons test, n = 3).

Article Snippet: Mouse monoclonal antibodies against cMyc (sc‐40, 1:2000 for IB, 1:200 for IHC, and 1:50 for ChIP), tubulin (sc‐5286, 1:2000 for IB), and CYP2B10 (sc‐73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

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

Journal: Advanced Science

Article Title: Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer

doi: 10.1002/advs.202205818

Figure Lengend Snippet: The PHGDH ACT domain is required for forming PHGDH/p300/cMyc/AF9 axis and regulates cMyc transactivation. 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 input 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 Escherichia 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) Chromatin immunoprecipitation (ChIP) analysis of cMyc enrichment on promoters of five canonical cMyc targets was performed using indicated cells. IgG was used as a blank control (mean ± SD, two‐tailed Student's t ‐test, n = 3). F) qRT‐PCR assay of the five genes from (E) (mean ± SD, two‐tailed Student's t ‐test, n = 3). G) 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. H) 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, p300, and PHGDH was performed using the indicated antibodies. NC, negative control. I) 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 the 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, 1:2000 for IB, 1:200 for IHC, and 1:50 for ChIP), tubulin (sc‐5286, 1:2000 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, Chromatin Immunoprecipitation, Two Tailed Test, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Advanced Science

Article Title: Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer

doi: 10.1002/advs.202205818

Figure Lengend Snippet: PHGDH/cMyc axis drives CXCL1/IL8 expression. 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) In PHGDH ‐depleted PLC/PRF/5 cells with rPHGDH‐WT or rPHGDH‐dACT or further depletion of c‐Myc, qRT‐PCR validated the top up‐regulated genes from (C) (mean ± SD, one‐way ANOVA followed by Dunnett's multiple comparisons test, n = 3). F) ELISA examined the concentration of CXCL1/IL8 and IL1B in the medium 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/IL8, 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 the cMyc‐associated complex. Immunoblotting analysis of PHGDH, p300, cMyc, and AF9 was performed using the indicated antibodies. I) qRT‐PCR validated CXCL1/IL8 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/IL8 and IL1B in the medium 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/IL8, PHGDH, and cMyc was performed using the indicated cells and antibodies. L) ChIP analysis of PHGDH, cMyc, p300, RNA Pol II (Pol 2), 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, 1:2000 for IB, 1:200 for IHC, and 1:50 for ChIP), tubulin (sc‐5286, 1:2000 for IB), and CYP2B10 (sc‐73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

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

Journal: Advanced Science

Article Title: Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer

doi: 10.1002/advs.202205818

Figure Lengend Snippet: Destruction of the PHGDH/cMyc axis hampered neutrophil/macrophage recruitment by liver cancer cells and impaired liver cancer progression. A) Cell proliferation was evaluated by spectrofluorometer at wavelength OD450 in PHGDH ‐depleted PLC/PRF/5 or Hep3B cells rescued with Vec, rPHGDH‐WT, rPHGDH‐NES, and rPHGDH‐dACT. Cells expressing shNT rescued with Vec were used as control (mean ± SD, two‐way ANOVA). B) Sorafenib inhibition was evaluated by trypan blue staining in PHGDH ‐depleted PLC/PRF/5 or Hep3B cells rescued with Vec, rPHGDH‐WT, rPHGDH‐NES, and rPHGDH‐dACT. Cells expressing shNT rescued with Vec were used as control (mean ± SD, two‐way ANOVA). C) Neutrophil recruitment was evaluated by cell migration, which was performed by placing the medium from culturing PHGDH ‐depleted PLC/PRF/5 or Hep3B cells in the lower well and neutrophil cells in the upper transwell chamber. Scale bars: 20 µm (mean ± SD, one‐way ANOVA followed by Tukey's multiple comparisons test, n = 3 per group). D) Macroscopic images and H&E staining of Phgdh LKO mouse livers under different AAV treatments (Phgdh‐WT with or without neutralized antibodies against Cxcl1/Il8 and Phgdh‐dACT) at W9 after hydrodynamic injection of MET/CAT constructs and the SB transposase. The tumor region is outlined with a white dashed line. Scale bars: 100 µm (upper); 1 cm (lower). The tumor nodule/liver weight ratios were measured (mean ± SD, one‐way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). E) Liver/body weight ratios of MET/CAT‐transfected mice under different AAV treatments (Phgdh‐WT with or without neutralizing antibodies (nAb) against Cxcl1/Il8 and Phgdh‐dACT) at W9 were measured (mean ± SD, one‐way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). F,H,J) IHC or IF staining of Ki67, Ly6G/CD11b, and F4/80 in mouse liver sections in (D). The relative number of Ki67 + cells (F), tumor‐associated neutrophil (TAN) (H), or tumor‐associated macrophage (TAM) (J) was pointed out by white arrows and calculated. Scale bars: 100 µm (F), 50 µm (H,J) (mean ± SD, one‐way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). G,I) Using fresh liver tumors in (D), representative flow cytometry data of neutrophils (CD11b + Ly6G + ) and TAMs (F4/80 + ) from indicated livers (mean ± SD, one‐way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). K) Kaplan–Meier plot showing the survival of MET/CAT‐transfected mice under different AAV treatments (Phgdh‐WT with or without neutralized antibodies against Cxcl1/Il8 and Phgdh‐dACT). (Log‐rank test, n = 5 per group). L) Using fresh liver tumors from the mice group Phgdh‐WT with or without neutralized antibodies against Ly6G, representative flow cytometry data of neutrophils (CD11b + Ly6G + ) from indicated livers (mean ± SD, one‐way ANOVA followed by Tukey's multiple comparisons test, n = 5 per group). M) Kaplan–Meier plot showing the survival of MET/CAT‐transfected mice under different treatments (Phgdh‐WT with or without neutralized antibodies against Ly6G). (Log‐rank test, n = 5 per group).

Article Snippet: Mouse monoclonal antibodies against cMyc (sc‐40, 1:2000 for IB, 1:200 for IHC, and 1:50 for ChIP), tubulin (sc‐5286, 1:2000 for IB), and CYP2B10 (sc‐73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

Techniques: Expressing, Control, Inhibition, Staining, Migration, Injection, Construct, Transfection, Flow Cytometry

Journal: Advanced Science

Article Title: Elevated Nuclear PHGDH Synergistically Functions with cMyc to Reshape the Immune Microenvironment of Liver Cancer

doi: 10.1002/advs.202205818

Figure Lengend Snippet: Nucleus‐localized PHGDH predicts poor prognosis and associates with cMyc in the clinic. 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 NATs using the two HCC proteomic datasets (Gao et al . 2019, Cell ; Jiang et al . 2019, Nature ) (Wilcoxon matched‐pairs signed rank test). C,D) 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. E) IHC staining of PHGDH in human liver cancer tissues. Representative images of the nucleus (Nuc), cytoplasm (Cyto), and both nucleus and cytoplasm (Nuc & Cyto) localized PHGDH were shown. Scale bars: 50 µm (left); 10 µm (right). F) 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). G) IF analyses of infiltrated neutrophils in human liver cancer tissues. H) The semi‐quantitative scoring between nuclear PHGDH intensity (using a scale from 0% to 100%) and neutrophils (using a scale from 0 to 300) was carried out (Pearson product‐moment correlation test). I) The positive correlation of PHGDH and cMyc or PHGDH and cMyc‐AcK148 in clinical liver cancer patients were examined by IHC. The right panels show the semi‐quantitative scoring (using a scale from 0 to 300) between two staining signals carried out (Pearson product‐moment correlation test). Scale bar, 50 µm. J) Survival plot of PHGDH expression, cMyc expression, the combined expression of PHGDH, and cMyc in TCGA‐LIHC data with median cutoff. (Log‐rank test.) K) Work model of the nonmetabolic role of PHGDH.

Article Snippet: Mouse monoclonal antibodies against cMyc (sc‐40, 1:2000 for IB, 1:200 for IHC, and 1:50 for ChIP), tubulin (sc‐5286, 1:2000 for IB), and CYP2B10 (sc‐73546, 1:50 for IHC) were purchased from Santa Cruz Biotech.

Techniques: Expressing, MANN-WHITNEY, Immunohistochemistry, Staining