Journal: Cell & Bioscience

Article Title: Modelling porcine NAFLD by deletion of leptin and defining the role of AMPK in hepatic fibrosis

doi: 10.1186/s13578-023-01124-1

Figure Lengend Snippet: Detection of oxidative stress in LEPTIN −/− pig livers. A Expression analysis of CYP2 family genes by real-time PCR. B. WB analysis and Immunofluorescence staining of CYP2E1. Scale bar represents 100 μm. C – E Detection of oxidative stress markers in the liver and serum. Content of MDA ( C ), NO ( D ) and SOD ( E ). N = 3/group. The bars represent the mean ± SD; *P < 0.05

Article Snippet: The total SOD activity detection kit (WST-8 method) (Beyotime, S-0101), lipid oxidation (MDA) assay kit (Beyotime, S-0131) and nitric oxide detection kit (Beyotime, S-0021) were utilized following the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining

Journal: Molecular Oncology

Article Title: PIKFYVE inhibitors trigger interleukin‐24‐dependent cell death of autophagy‐dependent melanoma

doi: 10.1002/1878-0261.13607

Figure Lengend Snippet: RNA‐seq analysis of genes upregulated by WX8 in melanoma A375 cells and HFF1 foreskin fibroblasts. (A) The total number of IL24 transcripts per 10 6 total transcripts [±SD for A375 ( n = 4) and HFF1 ( n = 3)] in cells treated for 24 h with either vehicle, 0.05 μ m or 1 μ m WX8. (B, C) RNA sequence analysis identified 37 936 different RNAs in A375 cells. The 100 most highly expressed genes were plotted against their RNA abundance. The top 5% of RNAs upregulated with 0.05 μ m WX8 were also present in the top 26% of RNAs upregulated with 1 μ m WX8. (D, E) RNA sequence analysis identified 31 249 different RNAs in HFF1 cells. Of these, 19 were upregulated by 0.05 μ m WX8, but 968 were upregulated by 1 μ m WX8. IL24 was upregulated in A375 cells, but not in HFF1 cells. Only PLA2G3, LIN00520, and INSIG1 were upregulated in both cell lines. Ratios < 1.5 were considered not significant. In panels B and D, the number of genes upregulated ≥ 1.5 [fold change] greater than vehicle treated cells is marked by horizontal broken lines. Panels B and C are the average of four samples. Panels D and E are the average of three samples.

Article Snippet: Cells were seeded at 50 000 cells per well in a 6‐well plate, and 16–18 h later treated with either vehicle or WX8 and then cultured for 72 h. The culture medium was then centrifuged at 18 000 g for 15 min at 4 °C to remove any detached cells, diluted and IL24 quantified using the IL24 ELISA Kit (OriGene, Rockville, MD, USA, EA100090) and manufacturer's instructions.

Techniques: RNA Sequencing, Sequencing

Journal: Molecular Oncology

Article Title: PIKFYVE inhibitors trigger interleukin‐24‐dependent cell death of autophagy‐dependent melanoma

doi: 10.1002/1878-0261.13607

Figure Lengend Snippet: WX8 triggered the EIF2AK3/PERK‐dependent ER‐stress response. The EIF2AK3/PERK‐dependent ER‐stress response in WX8‐treated melanoma A375 cells consisted of 17 genes that were detected by upregulation of their RNA and/or protein levels (Fig. ). (A) RNA levels were quantified by RNA‐seq analysis in both A375 cells and HFF1 cells. Gray bars are 0.05 μ m WX8. Black bars are 1 μ m WX8. Genes with RNA levels ≥ 1.5 fold change above vehicle (V)‐treated cells were considered as significant upregulation. (B) Upregulation of interleukin‐24 (IL24) RNA was detected by both RT‐PCR and RNA‐seq. (C) IL24 protein was detected by immunoblotting. Extracts from WX8‐treated cells were treated with PNGase F to remove N‐linked oligosaccharides from IL24. (D) Cells treated for 24 h with vehicle (V), 1 μ m WX8, 1 μ m thapsigargin (THAP) or 1 μ m tunicamycin (TUN) were immunoblotted for IL24 protein. (E) IL24 RNA in samples from panel D were quantified by RT‐PCR (V indicates ‘vehicle’). (F) Cells were treated with vehicle (V), 1 μ m WX8 or 10 μ m WX8 for 24 h. IL24 secreted into the culture medium by was quantified by ELISA assays. (G) Melanoma A375 cells were cultured with either vehicle or 1 μ m thapsigargin (THAP) for the indicated times. Whole cell extracts were then immuno‐blotted for the indicated proteins, which were identified by their molecular weight and their reaction with a specific antibody. Gene symbols are from the HUGO gene nomenclature committee. V indicates ‘vehicle’. Error bars indicate ± SEM for three samples. Statistical significance was P < 0.05 (*), P < 0.001 (***), P < 0.0001 (****). P > 0.05 was ‘not significant’ (ns). P ‐values in RNA‐seq were determined by Wald Chi‐square test using the DEseq2 platform. P ‐values of two data sets from the other experiments were calculated by Student's paired two‐tailed t ‐test using graphpad prism .

Article Snippet: Cells were seeded at 50 000 cells per well in a 6‐well plate, and 16–18 h later treated with either vehicle or WX8 and then cultured for 72 h. The culture medium was then centrifuged at 18 000 g for 15 min at 4 °C to remove any detached cells, diluted and IL24 quantified using the IL24 ELISA Kit (OriGene, Rockville, MD, USA, EA100090) and manufacturer's instructions.

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Molecular Weight, Two Tailed Test

Journal: Molecular Oncology

Article Title: PIKFYVE inhibitors trigger interleukin‐24‐dependent cell death of autophagy‐dependent melanoma

doi: 10.1002/1878-0261.13607

Figure Lengend Snippet: The EIF2AK3/PERK‐dependent ER‐stress response upregulated IL24 expression. (A) Melanoma A375 cells were cultured with either vehicle (V), 0.05 μ m WX8, or 1 μ m WX8 for either 24 or 48 h. Whole cell extracts were then immuno‐blotted for the indicated proteins, which were identified by their molecular weight and their reaction with a specific antibody. Thapsigargin (THAP) 1 μ m was included for comparison. (B) Cells were treated for 24 h with vehicle (V), 0.05 μ m WX8 or 1 μ m WX8 and the indicated proteins identified by immuno‐blotting from both cytoplasm and nuclear lysate. Lamin B1 (LMNB1) and Vinculin (VCL) were used as nuclear and cytoplasmic control proteins. (C) Cells were treated for 48 h with vehicle (V), 1 μ m WX8 or 1 μ m THAP, and either nontargeted siRNA (nt‐siRNA) or siRNA targeted against DDIT3 (siDDIT3). The indicated proteins were detected by immunoblotting whole cell extracts. (D) Cells were cultured for 24 h with increasing concentrations of WX8, and the indicated proteins (eIF2α, eIF2α‐P, and ACTB) identified by immunoblotting of whole cell extracts. (E) IL24 protein [both glycosylated and de‐glycosylated (PNGase F treated) were immunoblotted from samples as described in panel D]. (F) The ratios of IL24/ACTB and EIF2A‐P/ACTB were quantified by densitometry from the data in panels D and E. (G) The ratio of IL24 RNA relative to HFF1 IL24 RNA was determined by RT‐PCR in the indicated cell lines (± SEM, n = 3) and compared with cell viability, as previously quantified by their IC 50 for ATP loss . (H) The indicated cell lines were cultured for 24 h with vehicle, 0.05 μ m WX8 (gray bar) or 1 μ m WX8 (black bar). The ratios of IL24 RNA in WX8‐treated cells to vehicle‐treated cells were quantified by RT‐PCR (±SD, n = 2). Images in panels A through F are representative of three samples.

Article Snippet: Cells were seeded at 50 000 cells per well in a 6‐well plate, and 16–18 h later treated with either vehicle or WX8 and then cultured for 72 h. The culture medium was then centrifuged at 18 000 g for 15 min at 4 °C to remove any detached cells, diluted and IL24 quantified using the IL24 ELISA Kit (OriGene, Rockville, MD, USA, EA100090) and manufacturer's instructions.

Techniques: Expressing, Cell Culture, Molecular Weight, Comparison, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Oncology

Article Title: PIKFYVE inhibitors trigger interleukin‐24‐dependent cell death of autophagy‐dependent melanoma

doi: 10.1002/1878-0261.13607

Figure Lengend Snippet: IL24 upregulation was specific for PIKFYVE inhibition. (A) Melanoma A375 cells were cultured with the indicated PIKFYVE inhibitor (WX8, Apilimod/Apl, Vacuolin‐1/Vac, YM201636/YM) or autophagy inhibitor (Chloroquine/CQ) for 24 h. Expression of IL24 RNA relative to vehicle was quantified using RT‐PCR. (B) Example of an IL24 immunoblot from panel A in which IL24 protein is visible for all five inhibitors. (C) A375 cells were cultured for 24 h with the indicated inhibitors [WX8, Apl, CQ, Bafilomycin‐A1 (BafA1), Adriamycin (Adr), etoposide (Etop)]. The ratio of IL24 RNA in cells treated with inhibitor to RNA in cells treated with vehicle was quantified by RT‐PCR. (D) A375 cells were cultured for 24 h with the indicated concentrations of WX8, CQ and BafA1. The relative amounts of LC3‐II protein were detected by immunoblotting of whole cell extracts, thereby confirming that each inhibitor blocked autophagic flux to an equivalent extent. (E) A375 cells were cultured for 24 h with either vehicle (V), or the indicated concentration of WX8, Adr, or Etop. Equal amounts of total cell extract were subjected to immunoblotting for PARP, cleaved PARP (c‐PARP), γH2AX, and the cytoplasmic protein ACTB. Only Adr and Etop induced DNA damage, as indicated by the appearance of cPARP and γH2AX. (F) Phase contrast images of A375 cells treated with vehicle, apilimod, or NCT‐504 for 48 h at the indicated concentration. Bar indicates 20 μm. (G) Percentage of live (black) and dead A375 cells (gray) after treatment of vehicle, WX8, apilimod, and NCT‐504 for 72 h. (H) A375 cells are treated with indicated concentrations of WX8, apilimod, and NCT‐504 and IL24 protein was detected through immunoblotting. (I) A375 cells are treated with either nt‐siRNA or PIP4K2C siRNA for 24–72 h and cell lysates were immunoblotted for IL24. Error bars in panels A, C and G are ±SD, n = 2. Panels B, D–F, H, and I are representative of three samples.

Article Snippet: Cells were seeded at 50 000 cells per well in a 6‐well plate, and 16–18 h later treated with either vehicle or WX8 and then cultured for 72 h. The culture medium was then centrifuged at 18 000 g for 15 min at 4 °C to remove any detached cells, diluted and IL24 quantified using the IL24 ELISA Kit (OriGene, Rockville, MD, USA, EA100090) and manufacturer's instructions.

Techniques: Inhibition, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay

Journal: Molecular Oncology

Article Title: PIKFYVE inhibitors trigger interleukin‐24‐dependent cell death of autophagy‐dependent melanoma

doi: 10.1002/1878-0261.13607

Figure Lengend Snippet: WX8 disrupted autophagy and upregulated IL24 gene expression in melanoma A375 tumors. (A) Inbred female BALB/c nu/nu mice were inoculated on both flanks with melanoma A375 cells. Tumors were excised 21 days postinoculation. Bar indicates 1.5 cm. (B) When palpable tumors were evident (day 6), intraperitoneal injections of either vehicle (open circles), 20 mg WX8·kg −1 mouse weight (shaded circles), or 40 mg WX8·kg −1 mouse weight (solid circles) were administered each day (arrows) for 14 days. Tumor growth was inhibited in proportion to the WX8 concentration. Three mice were used for each treatment group. (C) Mouse body weights increased with time. (D) Tumors were excised at 21 days postinoculation and tumor sections were stained with hematoxylin and eosin. Vacuolation surrounding nuclei was evident in tumors from WX8‐treated mice but absent in vehicle‐treated mice. Bar indicates 50 μm. (E) Extracts from tumor slices revealed WX8‐dependent accumulation of LC3‐II, IL24, SQSTM1/p62, EIF2A, EIF2A‐P, ATF4, and DDIT3 proteins in tumors from WX8‐treated mice. ACTB and VCL are cytoplasmic proteins. Phosphorylated proteins are unstable in frozen samples. (F) SQSTM1 and LC3‐II proteins in panel E were quantified (±SD, n = 2). (G) SQSTM1 and IL24 RNA abundance from tumors from WX8‐treated mice relative to tumors in vehicle‐treated mice was quantified by RT‐PCR (± SEM, n = 3 mice or three tumors). Images in panels A, D, and E are representative of three samples.

Article Snippet: Cells were seeded at 50 000 cells per well in a 6‐well plate, and 16–18 h later treated with either vehicle or WX8 and then cultured for 72 h. The culture medium was then centrifuged at 18 000 g for 15 min at 4 °C to remove any detached cells, diluted and IL24 quantified using the IL24 ELISA Kit (OriGene, Rockville, MD, USA, EA100090) and manufacturer's instructions.

Techniques: Gene Expression, Concentration Assay, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Oncology

Article Title: PIKFYVE inhibitors trigger interleukin‐24‐dependent cell death of autophagy‐dependent melanoma

doi: 10.1002/1878-0261.13607

Figure Lengend Snippet: Ectopic expression of IL24 protein selectively induced melanoma cell death. (A) Melanoma A375 and foreskin fibroblasts Hs27 and HFF1 were infected for 72 h with the indicated number of plaques forming units (pfu) per mL of either AdV‐null or AdV‐ IL24 . Total cell extracts were then immunoblotted for IL24 protein. (B) The fraction of dead cells was determined at different pfu concentrations by staining one group with trypan blue and a separate group with propidium iodide. FACS analysis revealed the fraction of cells with < 2N DNA (Fig. ). (C) Phase contrast images of cells infected with either AdV‐null or AdV‐ IL24 with 5 × 10 5 pfu·mL −1 . Bar represents 20 μm. (D) Attached cells were collected by trypsinization, combined with unattached cells in culture medium, stained with trypan blue and then quantified as either live or dead for A375, Hs27 and HFF1 cell lines infected with either Adv‐Null or Adv‐ IL24 . (E) The indicated cell populations were subjected to FACS analysis to quantify the faction of cells with < 2N DNA content (Fig. ). A375 cells were also cultured with 0.4 μ m WX8 ± either AdV‐null or AdV‐ IL24 for comparison. Panels B, D, and E include ±SD, n = 2. Statistical significance was P < 0.05 (*), P < 0.01 (**), P < 0.0001 (****). P ‐values in RNA‐seq were determined by Wald Chi‐square test using the DEseq2 platform. P ‐values of two data sets from the other experiments were calculated by Student's paired two‐tailed t ‐test using graphpad prism . Images in panels A and C are representative of three samples.

Article Snippet: Cells were seeded at 50 000 cells per well in a 6‐well plate, and 16–18 h later treated with either vehicle or WX8 and then cultured for 72 h. The culture medium was then centrifuged at 18 000 g for 15 min at 4 °C to remove any detached cells, diluted and IL24 quantified using the IL24 ELISA Kit (OriGene, Rockville, MD, USA, EA100090) and manufacturer's instructions.

Techniques: Expressing, Infection, Staining, Cell Culture, Comparison, RNA Sequencing, Two Tailed Test

Journal: Molecular Oncology

Article Title: PIKFYVE inhibitors trigger interleukin‐24‐dependent cell death of autophagy‐dependent melanoma

doi: 10.1002/1878-0261.13607

Figure Lengend Snippet: Il24 gene expression is essential for WX8‐dependent cell death. Exon‐2 in the IL24 gene was ablated in melanoma A375 cells. (A) Surviving clones were isolated and screened for deletion within exon‐2 of IL24 gene and comparison with DNA size standards (std) using PCR technology. (B) Immunoblot of total cell extracts confirmed the absence of IL24 protein in A375( IL24 −/− ) clones. (C) WX8 upregulated expression of IL24 protein in wild‐type A375 cells but not in A375( IL24 −/− ) clones exemplified by c10 and c17. (D) Phase contrast images of A375 and A375( IL24 −/− ) cells. Bar indicates 20 μm. (E) ELISA assays confirmed the absence of IL24 protein in the cell culture medium of A375( IL24 −/− ) clones cultured for 24 h in the presence of either vehicle or 1 μ m WX8. (F) The fractions of live (impermeable to trypan blue) and dead (permeable to trypan blue) A375 and A375( IL24 −/− ) cells after culturing A375 wild‐type and A375( IL24 −/− ) clone 10 cells for 72 h with the indicated concentrations of WX8. (G) The fraction of cells with < 2N DNA content (less DNA than G1 phase cells) were considered dead. To allow a logarithmic axis, vehicle was plotted as 0.001 μ m WX8. Panels E–G include ±SD, n = 2. P ‐values in RNA‐seq were determined by Wald Chi‐square test using the DEseq2 platform. P ‐values of two data sets from the other experiments were calculated by Student's paired two‐tailed t ‐test using graphpad prism . Images in panels A through D are representative of three samples.

Article Snippet: Cells were seeded at 50 000 cells per well in a 6‐well plate, and 16–18 h later treated with either vehicle or WX8 and then cultured for 72 h. The culture medium was then centrifuged at 18 000 g for 15 min at 4 °C to remove any detached cells, diluted and IL24 quantified using the IL24 ELISA Kit (OriGene, Rockville, MD, USA, EA100090) and manufacturer's instructions.

Techniques: Gene Expression, Clone Assay, Isolation, Comparison, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, RNA Sequencing, Two Tailed Test