Journal: Frontiers in Immunology

Article Title: Identification of molecular subtypes based on PANoptosis-related genes and construction of a signature for predicting the prognosis and response to immunotherapy response in hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1218661

Figure Lengend Snippet: Ability of PANoptosis-related signature (PANRS) to predict immune landscape and immunotherapy response in hepatocellular carcinoma (HCC). (A) Differential tumor microenvironment (TME) between the high-risk and low-risk groups; (B) Comparative analysis of immune cell infiltration status between the high-risk and low-risk groups; (C–F) The correlation of risk scores with memory B cells (C) , M0 macrophages (D) , M1 macrophages (E) , and monocytes (F) ; (G) Differential expression of immune checkpoint molecules between the high-risk and low-risk groups; (H–K) Comparative analysis of tumor immune dysfunction and exclusion (TIDE) (H) , dysfunction (I) , exclusion (J) , and microsatellite instability (MSI) scores (K) between the high-risk and low-risk groups; (L) Analysis of the correlation of risk scores with complete response (CR)/partial response (PR) and stable disease (SD)/progressive disease (PD) in the IMvigor210 cohort; (M) Comparison of overall survival (OS) between the high-risk and low-risk group in the IMvigor210 cohort; (N, O) The predictive value of the expression of the signature genes LPCAT1 (N) and CBX2 (O) for immunotherapy response; (P) Effect of LPCAT1 expression levels on the OS of patients with bladder cancer; (Q) Distribution of tumor mutational burden (TMB) in different risk groups; (R) Differential expression of human leukocyte antigen (HLA) molecules in different risk groups. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following antibodies were used in western blotting analysis: anti- LPCAT1 antibody (1:500, Proteintech, 16112-1-AP), anti- CASP1 (1:6000, Proteintech, 22915-1-AP), anti- CASP3 (1:500, ABclonal, A2156), anti- CASP7 (1:600, Proteintech, 27155-1-AP), anti- CASP8 (1:500, ABclonal, A0215), anti- GSDME (1:5000, Proteintech, 13075-1-AP), anti-cleaved GSDME antibody(1:500, ABclonal, A23072), anti- GSDMD (Full Length+N terminal, 1:500, ABclonal, A20197), anti- MLKL (1:500, ABclonal, A21894), anti-phospho- MLKL antibody(1:50, ABclonal, A1244), and anti- GAPDH antibody (1:10000, Proteintech, 60004-1-Ig).

Techniques: Quantitative Proteomics, Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: Identification of molecular subtypes based on PANoptosis-related genes and construction of a signature for predicting the prognosis and response to immunotherapy response in hepatocellular carcinoma

doi: 10.3389/fimmu.2023.1218661

Figure Lengend Snippet: LPCAT1 knockdown inhibits hepatocellular carcinoma (HCC) cell proliferation and migration and promotes PANoptosis. (A) Quantitative real-time polymerase chain reaction analysis of LPCAT1 mRNA expression in healthy liver cell lines (THLE-2) and HCC cell lines (HCCLM3, MHCC97-H, and HepG2); (B) Western blotting analysis of LPCAT1 protein expression levels in healthy liver cell lines (THLE-2) and HCC cell lines (HCCLM3, MHCC97-H, and HepG2); GAPDH was used as an internal control; (C, D) The knockdown efficiency of different siRNA- LPCAT1 constructs in HepG2 and HCCLM3 cells was evaluated using western blotting; (E–H) Effects of transfection with siRNA– LPCAT1 #1, siRNA– LPCAT1 #2 or negative control (si-NC) on cell proliferation were assessed using ethynyl-deoxyuridine (EdU) and cell counting kit (CCK)-8 assays; (E, G) Representative images of the changes in the number of proliferating HepG2 and HCCLM3 cells in different groups after transfection and quantitative analysis of EdU-positive rate (original magnification, ×200; scale bar, 50 μm); (F, H) The line graphs show the changes in the viability of HepG2 and HCCLM3 cells in different groups at 0, 24, 48, and 72 h post-transfection; (I, J) Cell migration was examined using the wound-healing assay. Representative images and quantitative analysis of wound closure area in different groups at 0 and 36 h post-scratching are presented (original magnification, ×40; scale bar, 200 μm); (K) Transwell assay was used to evaluate the migration ability of transfected HCC cells. The upper panel shows representative images (original magnification, ×200; scale bar, 200 μm), while the lower panel (histogram) shows the number of migrated cells in different groups; (L-N) Immunoblotting analysis of (L) pro-CASP1 (P45) and activated (P20) CASP1, pro-GSDMD (-FL), and activated GSDMD (-N); and pro-GSDME (-FL) and activated (-N) GSDME; (M) pro-CASP3 (P35) and cleaved (P17) CASP3, pro-CASP7 (P35) and cleaved (P20) CASP7, and pro-CASP8 (P55) and cleaved (P18) CASP8; and (N) phosphorylated MLKL (pMLKL), and total MLKL (tMLKL) in HCCLM3 cells transfected with si-NC, si- LPCAT1 #1, and si- LPCAT1 #2. GAPDH was used as the internal control. Data are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The following antibodies were used in western blotting analysis: anti- LPCAT1 antibody (1:500, Proteintech, 16112-1-AP), anti- CASP1 (1:6000, Proteintech, 22915-1-AP), anti- CASP3 (1:500, ABclonal, A2156), anti- CASP7 (1:600, Proteintech, 27155-1-AP), anti- CASP8 (1:500, ABclonal, A0215), anti- GSDME (1:5000, Proteintech, 13075-1-AP), anti-cleaved GSDME antibody(1:500, ABclonal, A23072), anti- GSDMD (Full Length+N terminal, 1:500, ABclonal, A20197), anti- MLKL (1:500, ABclonal, A21894), anti-phospho- MLKL antibody(1:50, ABclonal, A1244), and anti- GAPDH antibody (1:10000, Proteintech, 60004-1-Ig).

Techniques: Knockdown, Migration, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Construct, Transfection, Negative Control, Cell Counting, CCK-8 Assay, Wound Healing Assay, Transwell Assay

Journal: PLOS Genetics

Article Title: LIPL-1 and LIPL-2 are TCER-1-regulated lysosomal lipases with distinct roles in immunity and fertility

doi: 10.1371/journal.pgen.1011804

Figure Lengend Snippet: A-C: Effects of lipl-1 (A) and lipl-2 (B) loss on broad neutral lipid (NL) and phospholipid (PL) populations in wild type (WT, black) and tcer-1 mutant (blue) animals. Bars indicate the fold-change of each data point normalized to the WT average. Statistical significance calculated using mixed-effects analysis with Tukey’s correction. C: Relative abundance of overall phosphatidylcholine (PC) levels. D: Categorization of NL (top, blue) and PL (bottom, green) species whose abundance is altered in tcer-1 mutants. For each class, number of species identified and those changed in tcer-1 mutants shown in the table. E: Relative abundance of PC 34:2 and PC 34:3 compared to all PCs in different strains. F-I: Distinct impacts of lipl-1 (F-H) and lipl-2 (I) on lipidome of tcer-1 mutants. F-H: Relative abundance of Cer 17:1;O2/22:0 (Cer 22) (F) , Cer 17:1;O2/24:0 (Cer 24) (G) and Cer 17:1;O2/26:0 (Cer 26) (H) compared to all ceramides. I: Relative abundance of lysophosphatidyl choline (LPC) 18:2 compared to all LPCs. Data derived from HPLC-MS/MS analysis of Day 1 young adult hermaphrodites isolated in 5–6 independent biological trials. Statistical significance was calculated using two-way ANOVA with Tukey’s correction. J-K: Ceramide supplementation enhances survival upon PA14 infection. Survival of wild type Ctrl (WT, black), lipl-1;tcer-1 ctrl (green), lipl-1;tcer-1 + Cer 24 (Olive), WT + Cer 24 (Red) raised on OP50 till Day1 and exposed to PA14. J: WT ctrl (m = 63.09 ± 1.53, n = 74/90), lipl-1;tcer-1 ctrl (m = 58.1 ± 1.33, n = 82/90), lipl-1;tcer-1 + Cer 24 (m = 73.74 ± 2.0, n = 71/90). K: WT ctrl (m = 63.09 ± 1.53, n = 74/90), WT + Cer 24 (m = 70.57 ± 1.78, n = 58/90). Data from additional PA14 survival trials in . Statistical significance was determined using log-rank Mantel Cox method and shown in each panel next to a given strain/condition. Asterisks indicate the statistical significance and their color the strain being used for the comparison. p ≤ 0.05(*), p < 0.01 (**), < 0.001 (***), < 0.0001 (****), n.s: not significant.

Article Snippet: Cer 24 ceramide (Avanti Polar Lipids, Cat. No. 860649P) and lysophosphatidylcholine 18:2 (LPC 18:2; MedChemExpress, Cat. No. HY-N9410) were dissolved in ethanol to prepare stock solutions at a concentration of 0.5 mg/mL.

Techniques: Mutagenesis, Derivative Assay, Tandem Mass Spectroscopy, Isolation, Infection, Comparison