Journal: Materials

Article Title: Improvement Properties of Hybrid Halide Perovskite Thin Films Prepared by Sequential Evaporation for Planar Solar Cells

doi: 10.3390/ma12091394

Figure Lengend Snippet: Scheme of the system used to grow CH 3 NH 3 PbI 3 (MAPI) or (NH 2 ) 2 CHPbI 3 (FAPI) films by sequential evaporation of precursors. PID: proportional integral derivative control algorithm.

Article Snippet: This includes the following units: High vacuum system, which allows to achieve a basis pressures around 2 × 10 −5 mbar Rotating substrate heating unit with facilities to control the temperature through a commercial PID system Knudsen cell-type evaporation sources heated through a thermocoax heating element.

Techniques: Evaporation, Control

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide impaired mitochondrial function and potently suppressed pSTAT3 signaling to regulate pathogenic tissue-resident memory cell differentiation and function in vitro (A) Mitochondrial ROS production in splenic immune cells and CD8 + T cells (stimulated with anti CD3 2 µg/ml, aCD28 2 µg/ml, IL-6 10ng/ml) was assessed after nano-sized graphene oxide (NGO) treatment using MitoSOX staining. (B) The mitochondrial oxygen consumption rate in splenic immune cells, under conditions mentioned above, was measured over time following NGO exposure. (C) Immunofluorescence imaging (200x) was performed to examine the distribution of splenic CD8 + T cells in the presence or absence of biotinylated NGO 10 µg/ml (CD8: green; NGO: white; DAPI: blue). (D) Western blotting analysis was used to evaluate phosphorylation of STAT3 at Ser727 and Tyr705, and STAT3 protein expression in NGO-treated splenic immune cells, stimulated under conditions mentioned above. (E – G) Flow cytometry was performed in splenic immune cells at same stimulation conditions to determine the frequencies of IFN-γ + and IL-17 + CD8 + CD103 + TRM-like T cells, as well as CD69 + CD103 + TRM cell subsets, in the presence or absence of NGO and regulatory T (Treg) cells. (H) Cytokine concentrations associated with psoriasis in splenic immune cell culture supernatants were measured by ELISA following treatment with 1 µg/ml NGO. (I) Multiplex immunofluorescence staining in splenic immune cells at same stimulation conditions was used to detect CD8 + CD103 + , CD69 + , pSTAT3 + , and IL-17 + cells, followed by quantitative analysis (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). n = number. All values are shown as the mean ± SD ( n = 6 for aCD3 + aCD28 + IL-6 condition, n = 6 for NGO 1 µg/ml, n = 6 for NGO 10 µg/ml). Each experiment was repeated three times. Statistical significance was determined by non-parametric Mann–Whitney U test for pairwise comparisons and one-way ANOVA using Kruskal-Wallis test for multiple group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: Cell Differentiation, In Vitro, Staining, Immunofluorescence, Imaging, Western Blot, Phospho-proteomics, Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Multiplex Assay, MANN-WHITNEY

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide (NGO) attenuated psoriatic skin inflammation by suppressing pSTAT3–IL-17 immune responses in vivo (A) Schematic overview of the experimental protocol. Psoriasis-like skin inflammation was induced in mice using imiquimod, followed by repeated NGO administration. (B) Measurement of spleen size and weight in control and NGO-treated mice. (C) Clinical scoring of skin scaling, erythema, thickness, and the Psoriasis Area and Severity Index (PASI) in control and NGO-treated mice. (D , G) Histological and immunohistochemical staining to determine epidermal thickness and Ki-67 + , CD8 + , IL-17 + , and pSTAT3 + cells in skin lesions. (E) Representative immunofluorescence images of spleen sections from each group show CD8 + T cells, biotin-labeled NGO, and nuclei in the spleen at magnifications of 200× and 400× (CD8: green; NGO: white; DAPI: blue). (F) Flow cytometric analysis of IFN-γ + and IL-17 + CD4 + and CD8 + T cells in the spleen. n = number. All data are shown as the mean ± SD ( n = 6 for SKG + IMQ, n = 8 for with NGO). Each experiment was repeated three times. Statistical significance was determined by two-way ANOVA with Bonferroni test in multiple groups and non-parametric Mann–Whitney U test for pairwise comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: In Vivo, Control, Immunohistochemical staining, Staining, Immunofluorescence, Labeling, MANN-WHITNEY

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide (NGO) attenuated psoriatic inflammation by inhibiting STAT3 activation and suppressing proinflammatory CD8 + tissue-resident memory (TRM) T cell accumulation in tissue (A) Western blotting analysis of splenic immune cells from psoriatic mice revealed that NGO treatment markedly inhibited phosphorylation of STAT3 at both Ser727 and Tyr705, quantified relative to total STAT3 and GAPDH ( n = 6 for SKG + IMQ, n = 6 for with NGO in western blot). (B) Flow cytometry showed that the frequency of activated CD69 + CD103 + CD8 + T cells, as well as IFN-γ + and IL-17 + CD103 + CD8 + T cell subsets, indicating suppression of key proinflammatory TRM cell populations in splenic immune cells from NGO treated psoriatic mice. (C) Multiplex immunofluorescence imaging and quantification demonstrated significant decreases in CD8 + CD103 + CD69 + TRM cells, CD8 + CD103 + pSTAT3 + , and CD8 + CD103 + IL-17 + cells in spleen tissues after NGO treatment (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). (D) Similar immunofluorescence analyses of skin lesions supported reduced presence of TRM cell markers and effector cytokines with NGO treatment, confirmed by quantitative positive cells counting (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). n = number. All data are shown as the mean ± SD ( n = 6 for SKG + IMQ, n = 8 for with NGO in flow cytometry and tissue data). Each experiment was repeated three times. Statistical significance was determined by non-parametric Mann–Whitney U test for pairwise comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Flow Cytometry, Multiplex Assay, Immunofluorescence, Imaging, MANN-WHITNEY

Journal: Journal of Nanobiotechnology

Article Title: NGO ameliorates psoriasis by modulating mitochondrial function and suppressing pSTAT3–IL-17–expressing CD8 + TRM cells

doi: 10.1186/s12951-025-04020-7

Figure Lengend Snippet: Nano-sized graphene oxide (NGO) restored mitochondrial function and suppressed proinflammatory tissue-resident memory CD8 + T cells in the peripheral blood of patients with psoriasis by regulating the pSTAT3–IL-17 axis. (A, B) Mitochondrial ROS (MitoSOX staining) was measured in whole peripheral blood mononuclear cells (PBMCs) and CD8 + CD103 + T cells from patients with psoriasis following NGO treatment, highlighting altered mitochondrial function. (C) The mitochondrial oxygen consumption rate was analyzed in healthy control PBMCs under conditions for psoriatic IL-17–expressing T cell differentiation (stimulated with anti CD3 2μg/ml, aCD28 2μg/ml, IL-23 10ng/ml). (D) Immunofluorescence imaging showed the co-localization and frequency of CD8 + T cells in the presence or absence of NGO 10μg/ml (CD8: green; NGO: white; DAPI: blue), with marked reduction upon NGO treatment in psoriasis patients’ PBMCs. (E) Western blotting and quantification demonstrated significant inhibition of STAT3 phosphorylation (Ser727 and Tyr705) in psoriasis patients’ PBMCs treated with NGO 10μg/ml. (F, G) Flow cytometric analysis showed that the population of IFN-γ + or IL-17 + CD4 + /CD8 + T cells and CD8 + CD103 + tissue-resident memory (TRM)-like T cells, as well as their cytokine production in psoriasis patients’ PBMCs. (H) The frequency of CD8 + CD69 + CD103 + and IFN-γ + or IL-17 + TRM cell subsets, as determined by flow cytometry in psoriasis patients’ PBMCs. (I) Regulatory T cell (Treg, Foxp3 + CD25 + CD4 + ) frequency assessed by flow cytometry in psoriasis patients’ PBMCs. (J) Multiplex immunofluorescence (200ⅹ) of patient-derived immune cells further confirmed decreased TRM cell marker CD8 + CD103 + , CD69 + , pSTAT3 + , and IL-17 + expression after NGO treatment, and quantitative analysis supported significant reductions in psoriasis patients’ PBMCs (CD8: green; CD103: red; CD69, pSTAT3, IL-17: white; DAPI: blue). Ps: psoriatic, HC: healthy control. n = number. All data are shown as the mean ± SD (n=6 for Psoriasis PBMC, n=6 for with NGO 10 μg/ml). Detailed patient information is described in the supplementary materials. Each experiment was repeated two times. Statistical significance was determined by non-parametric Mann–Whitney U test for pairwise comparisons. * P < 0.05,** P < 0.01, *** P < 0.001,**** P < 0.0001.

Article Snippet: After blocking, the membranes were incubated at room temperature for 35 min with primary antibodies against pSTAT3 (Ser727) (#9134; Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (Tyr705) (#9131; Cell Signaling Technology), STAT3 (#9139; Cell Signaling Technology), and GAPDH (ab181602; Abcam), all diluted in 0.4% skim milk in 1× Tris-buffered saline with 0.1% Tween-20 (TBS-T).

Techniques: Staining, Control, Expressing, Cell Differentiation, Immunofluorescence, Imaging, Western Blot, Inhibition, Phospho-proteomics, Flow Cytometry, Multiplex Assay, Derivative Assay, Marker, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 1. APOE accumulates in the MAMs of cultured hepatocytes, equally evident during APOE3 and APOE4 overexpression: (a) subcellular fractions were isolated from Huh7 cells by Percoll density gradient ultracentrifugation and analyzed by Western blotting. Representative images show the accumulation of the APOE protein in pure MAMs. Respective marker proteins were detected to visualize the purity of mitochondria (COX), MAMs (CANX) and cytosol (TUB); (b) APOE protein levels were quantified in the whole cell sample and crude and pure MAM fractions and normalized to the APOE level in the whole cell sample. In the pure MAM, the APOE protein level was significantly higher compared to the whole cell (p < 0.05, unpaired t-test) as indicated by an asterisk (*). The data are means ± SEM (n = 3); (c) no APOE isoform-dependent difference was observed in the accumulation of APOE in the pure MAM fraction in APOE3- and APOE4-transfected Huh7 cells. Data are means ± SEM (n = 2) and the accumulation of APOE in MAMs is related to the APOE protein level in the whole cell samples; subcellular fractions: c. mito., crude mitochondria; p. mito., pure mitochondria; c. MAM, crude mitochondria ER-membranes (MAMs), p. MAM, pure MAM.

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Over Expression, Isolation, Western Blot, Marker, Transfection

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 2. The number of visualized MERCs in cultured hepatocytes and MAM-assembling protein levels in the mouse liver are similar in presence of APOE3 and APOE4: (a) representative images of MERC-PLA experiments in APOE-transfected Huh7 cells that were incubated with 1 or 5 g/L glucose for six hours. VDAC1-IP3R1 PLA signals are shown in green, APOE was additionally stained in red to identify successfully transfected cells, and cell nuclei appeared blue by staining with DAPI. 400× magnification; scale bar 5 µm; (b) PLA signals from at least 100 cells per sample from three independent experiments were quantified showing a significant reduction of PLA signals per cell in all samples in response to the glucose challenge. No difference was observed comparing APOE3-, APOE4- and mock- transfected cells. Significance was accepted at p < 0.05, indicated with an asterisk (*); a two-way ANOVA was performed followed by the Šídák’s multiple comparisons test; (c) MAM-assembling as well as ER and mitochondrial marker proteins were analyzed in the livers of APOE-targeted replacement mice by Western blotting and representative images are shown; (d) densitometric analysis revealed no significant differences between APOE3 and APOE4 mice (unpaired t-test). Target band intensity was normalized by total protein load and related to the mean of APOE3 mice. Data are means ± SEM (n = 5–6).

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Transfection, Incubation, Staining, Marker, Western Blot

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 3. Mitochondrial APOE-interacting proteins are remotely connected to MAMs, with no apparent difference comparing APOE3- and APOE4-transfected cells. (a) Visualization of the eleven higher abundant protein–protein interactions (PPIs) shared by APOE3- and APOE4-transfected cells compared to unmodified Huh7 cells. PPIs were categorized by their main cellular localization identified according to The Human Gene Database GeneCards. Hitherto unknown PPIs (7/11) are highlighted by the greater thickness and blue color of the borderline. (b) Representative Western blot images of the presence of the APOE-interacting proteins LONP1, TOMM40 and VDAC1 in subcellular fractions isolated from Huh7 cells. Respective marker proteins for mitochondrial and ER-related MAM proteins were detected (GRP75, mitochondria, MAM; and PEMT, ER and MAM). (c) Representative Western blot images showing APOE, LONP1, TOMM40 and VDAC1 as well as the ER/MAM marker CANX in subcellular fractions of APOE3- and APOE4-transfected cells. (d) Target band intensities were quantified and normalized by total protein load per lane. The target protein level in the pure MAM fraction was related to the whole cell sample showing similar results in APOE3- and APOE4-transfected cells. Data are means ± SEM (n = 2). Subcellular fractions: c. mito., crude mitochondria; p. mito., pure mitochondria; c. MAM, crude mitochondria ER membranes (MAMs), p. MAM, pure MAM.

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Transfection, Protein-Protein interactions, Western Blot, Isolation, Marker

Journal: International journal of molecular sciences

Article Title: Readdressing the Localization of Apolipoprotein E (APOE) in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes (MAMs): An Investigation of the Hepatic Protein-Protein Interactions of APOE with the Mitochondrial Proteins Lon Protease (LONP1), Mitochondrial Import Receptor Subunit TOM40 (TOMM40) and Voltage-Dependent Anion-Selective Channel 1 (VDAC1).

doi: 10.3390/ijms251910597

Figure Lengend Snippet: Figure 4. Presence in MAMs and extent of the PPI of selected candidates and APOE in ER-stressed cultured hepatocytes. (a) Western blot images of LONP1, TOMM40, VDAC1 and APOE detection in subcellular fractions of unmodified Huh7 cells. Thapsigargin treatment (50 µM, 24 h) provoked the accumulation of LONP1 and APOE in MAMs relative to the whole cell sample and compared to untreated control cells. GRP75 served as a marker for MAMs and the positive control for thapsigargin induced MAM protein translocation. c. MAM, crude mitochondria ER membranes (MAMs), p. MAM, pure MAM; (b) representative Western blot images of LONP1, TOMM40 and VADC1 in APOE co-IP samples from unmodified Huh7 cells treated with thapsigargin (50 µM, 24 h). No signals were visible in the IP negative control (no antibody used, no AB). S, IP supernatant, inp., input control; (c) target band intensity was normalized by the corresponding APOE band intensity. Relative target protein levels in thapsigargin-stressed cells were related to the mean of untreated cells (expressed as thapsigargin-induced change). Data are means from three individual cell culture experiments and co-IP (n = 3).

Article Snippet: Then, the cells were incubated with ligase (30 min, 37 ◦C), polymerase (100 min, 37 ◦C) and fluorescent anti-APOE antibody (1:20, 90 min, room temperature; sc-13521 AF546, Santa Cruz, Dallas, TX, USA).

Techniques: Cell Culture, Western Blot, Control, Marker, Positive Control, Translocation Assay, Co-Immunoprecipitation Assay, Negative Control

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