Journal: Scientific Reports

Article Title: Combining DNMT and HDAC6 inhibitors increases anti-tumor immune signaling and decreases tumor burden in ovarian cancer

doi: 10.1038/s41598-020-60409-4

Figure Lengend Snippet: DNMT1 protein levels are decreased by combination treatment of DNMTi and HDAC6i. ( A ) Ovarian cancer cell lines were treated as in Fig. and protein was extracted at Day 7 after treatment with IFN-gamma (IFN-γ+) (to assess MHC I and PD-L1 expression, in later figures) or control (IFN-γ -). Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( B ) The TykNu cell line was treated as in ( A ) and the protein synthesis cycloheximide added to cells on Day 7 for 0, 4, and 8 hours at 10 μM as indicated on the blot. Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( C ) Stable knockdowns of the HDAC6 protein were generated in the ID8 Trp53+/+ and Trp53−/− cell lines . Protein was extracted and immunoblots were run for the DNMT1 protein with B-actin as a loading control. Immunoblot membranes were probed for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( D ) Immunoblot showing knockdown of HDAC6 protein with a-Tubulin as a loading control. Protein was extracted and immunoblots were run for the HDAC6 protein with B-actin as a loading control. Immunoblots were probed for HDAC6 (131 kDa) and tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( E ) Ovarian cancer cell lines were treated as in Fig. and RNA was extracted at Day 7. qRT-PCR was run for DNMT1, DNMT3a, and DNMT3b and TBP was used as a reference gene. *p < 0.05 compared to Mock.

Article Snippet: The antibodies used for immunoblotting included: DNMT1 (Sigma, D4692), PD-L1 (ProSci, 4059), HDAC1 (Cell Signaling, 2062), HDAC2 (Cell Signaling, 2540), HDAC6 (Assay Biotech, C0026), acetyl-alpha Tubulin (Cell Signaling, 3971), alpha-Tubulin (Cell Signaling, 3873).

Techniques: Expressing, Control, Isolation, Western Blot, Generated, Knockdown, Quantitative RT-PCR

Journal: Cancer Communications

Article Title: NAT10 regulates tumor progression and immune microenvironment in pancreatic ductal adenocarcinoma via the N4‐acetylated LAMB3‐mediated FAK/ERK pathway

doi: 10.1002/cac2.70045

Figure Lengend Snippet: NAT10 was associated with CD8 + T cell exhaustion in the PDAC TME. (A) Cell subset plot showing 10 subsets of T_NK cells in subcutaneous tumors collected from negative control (NC), Nat10 knockdown (sh Nat10 ‐1), and Nat10 overexpression ( Nat10 ‐OE) groups, as analyzed by the UMAP dimensionality reduction algorithm. (B) Histogram showing the proportions of 10 subsets of T_NK cells in sh Nat10 ‐1, NC, and Nat10 ‐OE groups. The red rectangle indicates the predominant abundance of CD8 + Tex (type I) subset in the Nat10‐ OE group. (C) Dot plot showing the mean expression and expression proportions of marker genes and functional signatures of the nine distinct T cell subsets. (D) Representative micrographs of immunofluorescence staining of CD8a (red) showing the proportion of CD8 + T cells in the subcutaneous tumor sections from sh Nat10 ‐1, NC, and Nat10 ‐OE groups ( n = 3 per group). Cell nuclei were counterstained with DAPI (blue). The quantification results are shown below. (E) Representative micrographs of multiplexed immunofluorescence staining of CD8a (red), PD‐1 (yellow), IFNg (orange), and Prf1 (green) showing the proportion of CD8 + Tex (type I) subset in the subcutaneous tumor sections from sh Nat10 ‐1, NC, and Nat10 ‐OE groups ( n = 3 per group). Cell nuclei were counterstained with DAPI (blue). The quantification results are shown below. Abbreviations: Bcl2, B‐cell lymphoma‐2; CD4 + Tex, exhausted CD4 + T cells; CD4 + Tn, naïve CD4 + T cells; CD8 + Tc, cytotoxic CD8 + T cells; CD8 + Tex, exhausted CD8 + T cells; CD8 + Tn, naïve CD8 + T cells; Ctla4, ctlantigen‐4; DAPI, 4',6‐Diamidino‐2‐phenylindole; γδT, gamma‐delta T cells; IFNg/Ifng, interferon gamma; Lag3, lymphocyte‐activation gene 3; NAT10, N‐acetyltransferase 10; NC, negative control; NK, natural killer cells; ns, not significant; OE, overexpression; PD‐1, programmed cell death 1; PDAC, pancreatic ductal adenocarcinoma; Pdcd1, programmed cell death 1; Prf1, perforin; Tgfb1, transforming growth factor beta 1; TME, tumor microenvironment; T_NK cells, T cells and NK cells; Treg, regulatory T cells; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: The cells were fixed in 4% PFA at room temperature for 15 minutes and incubated with recombinant human/mouse PD‐1 Fc chimeric protein (MedChemExpress) at room temperature for 1 hour, followed by incubation with Alexa Fluor TM 647‐conjugated secondary antibodies (Invitrogen) at room temperature for 1 hour.

Techniques: Negative Control, Knockdown, Over Expression, Expressing, Marker, Functional Assay, Immunofluorescence, Staining, Activation Assay

Journal: Cancer Communications

Article Title: NAT10 regulates tumor progression and immune microenvironment in pancreatic ductal adenocarcinoma via the N4‐acetylated LAMB3‐mediated FAK/ERK pathway

doi: 10.1002/cac2.70045

Figure Lengend Snippet: NAT10 may regulate CD8 + T cell function via PD‐1/PD‐L1 axis in PDAC. (A) Representative micrographs of immunofluorescence staining of PD‐1 (red) showing PD‐1/PD‐L1 binding on PDAC cells in negative control (NC) and NAT10 / Nat10 overexpression ( NAT10/Nat10 ‐OE) groups, with and without Naamidine J (10 µmol/L, 24 hours) treatment ( n = 3 per group). Cell nuclei were counterstained with DAPI (blue). The quantification results are shown on the right. (B) Representative results of Western blotting showing PD‐L1 expression in PDAC cells, including NC and NAT10 / Nat10 ‐OE groups, with and without Naamidine J (10 µmol/L, 24 hours) treatment ( n = 3 per group). GAPDH was used as a loading control. The numbers below the bands indicate the relative band density quantified by ImageJ, normalized to GAPDH. (C) Representative results of CD8 + T cell‐mediated tumor cell‐killing assay in NC and NAT10 ‐OE groups, with and without Naamidine J (10 µmol/L, 24 hours) treatment ( n = 3 per group). The quantification results are shown below. (D) Schematic showing the schedule of Naamidine J treatment in the animal models bearing subcutaneous tumors. PanO2 cells of negative control (NC) and Nat10 overexpression ( Nat10 ‐OE) were subcutaneously injected in the flanks of mice on day 0. Naamidine J was intraperitoneally injected into mice on days 28, 31, 34, 37, 40, 43, 46, 49, 52 (30 mg/kg, once every 3 days), and an equal volume of PBS was intraperitoneally injected accordingly as a control. The tumors were harvested on day 56 for analysis. (E) Growth curve of subcutaneous tumor size from the NC and Nat10 ‐OE groups, with PBS or Naamidine J treatment ( n = 5 per group). (F) Quantification results showing subcutaneous tumor size on day 56 in the NC and Nat10‐ OE groups, with PBS or Naamidine J treatment (n = 5 per group). (G) Quantification results showing the inhibitory efficacy of Naamidine J treatment on tumor size in the NC and Nat10‐ OE groups ( n = 5 per group). Inhibitory efficacy in the NC group = [tumor size NC + PBS – tumor size NC + Naamidine J ] / tumor size NC + PBS × 100%, and inhibitory efficacy in the Nat10 ‐OE group = [tumor size Nat10 ‐OE + PBS – tumor size Nat10 ‐OE + Naamidine J ] /tumor size Nat10 ‐OE + PBS × 100%. (H) Representative results of flow cytometry showing the proportion of infiltrating PD‐1 + IFNg + /PD‐1 − IFNg + CD8 + T cells from the subcutaneous tumors in the NC and Nat10‐ OE groups, with PBS or Naamidine J treatment ( n = 5 per group). The quantification results are shown below. (I) Representative results of flow cytometry showing the proportion of infiltrating PD‐1 + Prf1 + /PD‐1 − Prf1 + CD8 + T cells from the subcutaneous tumors in the NC and Nat10‐ OE groups, with PBS or Naamidine J treatment ( n = 5 per group). The quantification results are shown below. Abbreviations: CD8 + Tex, exhausted CD8 + T cells; CD8 + Tc, cytotoxic CD8 + T cells; DAPI, 4',6‐Diamidino‐2‐phenylindole; ‌‌GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IFNg, interferon gamma; PBS, phosphate buffered saline; PDAC, pancreatic ductal adenocarcinoma; PD‐1, programmed cell death 1; PD‐L1, programmed cell death ligand 1; Prf1, perforin; NAT10, N‐acetyltransferase 10; NC, negative control; ns, not significant; OE, overexpression.

Article Snippet: The cells were fixed in 4% PFA at room temperature for 15 minutes and incubated with recombinant human/mouse PD‐1 Fc chimeric protein (MedChemExpress) at room temperature for 1 hour, followed by incubation with Alexa Fluor TM 647‐conjugated secondary antibodies (Invitrogen) at room temperature for 1 hour.

Techniques: Cell Function Assay, Immunofluorescence, Staining, Binding Assay, Negative Control, Over Expression, Western Blot, Expressing, Control, Injection, Flow Cytometry, Saline

Journal: Cancer Communications

Article Title: NAT10 regulates tumor progression and immune microenvironment in pancreatic ductal adenocarcinoma via the N4‐acetylated LAMB3‐mediated FAK/ERK pathway

doi: 10.1002/cac2.70045

Figure Lengend Snippet: Graphic illustration on the role and mechanism of NAT10 in regulating tumor progression and immune microenvironment in PDAC. NAT10 promotes the malignant progression of PDAC cells by enhancing LAMB3 mRNA stability via ac4C modification, thereby activating the FAK/ERK pathway. In addition, NAT10 upregulates PD‐L1 expression in tumor cells through the LAMB3/FAK/ERK pathway and is associated with the abundance of intermediate exhausted CD8 + T cells in the PDAC TME. Blocking of PD‐1/PD‐L1 axis with Naamidine J increases the proportion of cytotoxic CD8 + T cells and decreases the proportion of intermediate exhausted CD8 + T cells in the PDAC TME, thereby inhibiting tumor malignant progression. Abbreviations: ac4C, N4‐acetylcytidine; ERK, extracellular regulated protein kinases; FAK, focal adhesion kinase; IFNg, interferon gamma; NAT10, N‐acetyltransferase 10; PDAC, pancreatic ductal adenocarcinoma; PD‐1, programmed cell death 1; PD‐L1, programmed cell death ligand 1; p‐ERK, phosphorylated extracellular regulated protein kinases; p‐FAK, phosphorylated focal adhesion kinase; Prf1, perforin; p‐Src, phosphorylated sarcoma; Src, sarcoma; TME, tumor microenvironment.

Article Snippet: The cells were fixed in 4% PFA at room temperature for 15 minutes and incubated with recombinant human/mouse PD‐1 Fc chimeric protein (MedChemExpress) at room temperature for 1 hour, followed by incubation with Alexa Fluor TM 647‐conjugated secondary antibodies (Invitrogen) at room temperature for 1 hour.

Techniques: Modification, Expressing, Blocking Assay

Journal: bioRxiv

Article Title: PDCD1 expression increases at elevated temperatures

doi: 10.1101/2025.05.22.652424

Figure Lengend Snippet: Up-regulation of PDCD1 transcription after heat shock in human cell lines and mouse tissues can be mediated by HSF1. ( A ) PDCD1 and HSPA1A (positive control for the heat shock response) transcript levels after heat shock (HS) were analyzed by RT-qPCR in human cell lines. ( B ) Pdcd1 and Hspa1 expression after heat shock (HS in vivo ) was analyzed by RT-qPCR in the mouse thymus, spleen, and heart. The readings were normalized against the mean of reference genes and presented versus Ctr in the spleen. Statistical significance versus corresponding Ctr is shown in the tables below. ( C ) HSF1 binding to the PDCD1 promoter was analyzed by ChIP-qPCR in the HL-60 cell line, untreated (Ctr) and after heat shock (HS at 43 °C). HSF1 binding to the negative locus and HSPA1A promoter were shown as negative and positive controls, respectively. ( D ) Western blot analysis of HSF1 levels in HL-60 cells: wild type (WT) and individual clones obtained after CRISPR/Cas9 editing (with HSF1 deficiency: #49, #215, #16, and unaltered: #18). ( E ) HSF1 binding to the PDCD1 promoter (analyzed by ChIP-qPCR) confirming the absence of heat-induced (HS at 43 °C) binding in the HSF1-deficient clone (#16). HSF1 binding to the negative locus and HSPA1A promoter were shown as negative and positive controls, respectively. ( F ) RT-qPCR analysis of PDCD1 and HSPA1A levels in WT and modified HL-60 cells. *** p < 0.0001, ** p < 0.001, * p < 0.05 (significance of differences).

Article Snippet: The cell pellet was resuspended in PBS, and CoraLite® Plus 488-conjugated antibodies were added: 0.4 μg anti-PDCD1 (Proteintech, Cat# CL488-66220; RRID:AB_2883287) or 0.4 μg mouse IgG1 isotype control (Proteintech, Cat# CL488-66360-1; RRID:AB_2934458).

Techniques: Positive Control, Quantitative RT-PCR, Expressing, In Vivo, Binding Assay, ChIP-qPCR, Western Blot, Clone Assay, CRISPR, Modification

Journal: bioRxiv

Article Title: PDCD1 expression increases at elevated temperatures

doi: 10.1101/2025.05.22.652424

Figure Lengend Snippet: Heat shock treatment can increase PDCD1 protein levels which may have functional consequences. ( A ) Western blot analyses in human cell lines and mouse lymph nodes heat-shocked (HS, 1 h at 43 °C; 42.5 °C in the case of NK-92 and 42 °C – mouse tissues) with indicated recovery time. HSPA1 and ACTB or GAPDH were used as positive controls for the HS response and loading controls, respectively. Graphs show the results of densitometric analyses (in the case of mouse tissues, the glycosylated form was not analyzed due to the overlap of the IgG signal detected by the secondary antibody). ( B ) Cell surface staining of PDCD1 was analyzed by flow cytometry in untreated (Ctr) and heat-shocked (HS, 1h at 43 °C and 12h recovery) cells. The counts (y-axis not to scale) from the blue squares areas are shown (as % of parent) in the table on the right. ( C ) Cytotoxicity of untreated and heat shocked NK-92 cells against T47D and CAL120 cells. The absorbance ratio: absorbance of the crystal violet stained target cells after 24 h co-culture with NK-92 was normalized versus absorbance of unattacked cells which is 1.0 (red dashed line). Boxplots represent the median, upper and lower quartiles, maximum and minimum. *** p < 0.0001, ** p < 0.001, *p<0.05 (significance of differences).

Article Snippet: The cell pellet was resuspended in PBS, and CoraLite® Plus 488-conjugated antibodies were added: 0.4 μg anti-PDCD1 (Proteintech, Cat# CL488-66220; RRID:AB_2883287) or 0.4 μg mouse IgG1 isotype control (Proteintech, Cat# CL488-66360-1; RRID:AB_2934458).

Techniques: Functional Assay, Western Blot, Staining, Flow Cytometry, Co-Culture Assay