Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Strategy for CRISPR Cas9 mediated genetic depletion of Cpt1a in primary BMDMs. (B) Seahorse analysis of OCR in Cpt1a KO and control BMDMs on DMXAA stimulation on sequential treatment with oligomycin, FCCP and Rotenone/Antimycin A (n=8 per group). (C-D) Histograms of basal OCR (C) and maximal respiration (D) of seahorse analysis in B . (E) Histograms showing IFN-β cytokine levels in Cpt1a KO and control BMDMs (n=4 per group) on treatment with DMXAA for indicated time points (0-6h). Results measured were normalized to the cell counts in each corresponding wells using two-way ANOVA followed by Tukey’s multiple comparisons test. (F) Representative immunoblots of pSTAT1, STAT1, p-STAT2, STAT2, ISG15, p-IRF3, IRF3 and vinculin in DMXAA treated control and Cpt1a KO BMDMs upon treatment with DMXAA for indicated time points (0-6h) (n=3 per group). (G) Representative blots of Ac-His3 K9/K14, total His3 and vinculin in control and Cpt1a KO BMDMs upon treatment with DMXAA for indicated time points (0-6h) (n=3 per group). (H) Schematic of proposed epigenetic control whereby type 1 IFN in a feed forward manner increases further interferon production in response by increaing myeloid cell mitochondrial FAO to generate Acetyl-CoA and histone acetylation. Asterisks represent - * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: CRISPR, Control, Western Blot

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Schematic of the murine infection study design. LCMV infected C57bl/6 mice were sacrificed at indicated time points (0,1,3,5 and 8 days) post infection and splenocytes were isolated (n=5 mice per group/day). CD11b+ myeloid cells were separated from the splenocytes and used for this study. (B-G) Histograms showing quantitative RT-PCR analysis of type 1 IFN response genes Ifnb (B) , Stat1 (C) , Stat2 (D) , Isg15 (E) and mitochondrial FAO enzymes Acat1 (F) and Cpt1a (G) in CD11b+ myeloid cells isolated from the splenocytes of infected mice. Data were normalized to 18 S rRNA and represented as means ± SEM. Splenocytes from infected mice were subjected to treatment with 5µM FaO Blue followed by staining with PE conjugated CD11b+ antibody. (H) Representative data of flow cytometry based measurement of fluorescence intensity of FaO Blue in CD11b+ splenocytes from infected mice at indicated time points (J) Representation of flow cytometric geometric mean fluorescence intensity of FaO Blue in infected mice at different timepoints. (K) Representative microscopic images of CD11b+ myeloid cells, isolated from the splenocytes of infected mice at different timepoints, incubated with FaO Blue. (I) Histograms of fluorescence intensity of FaO Blue from microscopic images in I . Data represented as means ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test. Asterisks represent * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: Infection, Isolation, Quantitative RT-PCR, Staining, Flow Cytometry, Fluorescence, Incubation

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Representative immunoblots of p-STAT1, STAT1, p-STAT2, STAT2, p-IRF3, IRF3 and vinculin from control and Isg15 KO BMDMs stimulated with DMXAA for different timepoints (0-12h).(n=5) (B) Volcano plot representation of differentially expressed genes in IFN-α treated PBMCs from ISG15 deficient patients compared to IFN-α treated PBMCs from healthy volunteers (GSE60359). x-axis represents log2 fold change in gene expression, and y-axis represents the −log10 adjusted p-value. Each point corresponds to a gene. Genes with large fold changes and high statistical significance appear toward the top left and top right of the plot, highlighting significantly downregulated and upregulated genes, respectively. Genes associated with type 1 IFN response has been highlighted as yellow dots. (C-E) Dot plot representation of scRNA analysis to understand the expression pattern of FAO associated genes in different immune cells from dermal tissues of healthy volunteers (C) , DLE (D) and SLE (E) patients. Dot size represents the percentage of immune cells expressing each gene and dot color represents the average expression level of indicated genes. (F) Schematic of the clinical validation study to determine FAO in CD14+ myeloid cells from PBMCs of control vs. SLE patients. PBMCs isolated from blood of 9 SLE patients and age matched healthy volunteers were incubated with 5 µM FaO Blue for 1h followed by staining with APC conjugated anti CD14 antibody. The CD14+ populations were gated and analyzed for fluorescent intensity of FaO blue by flow cytometry. (G) Representative data of flow cytometry based measurement of FaO Blue intensity in CD14+ gated PBMCs from SLE patient and respective age matched healthy volunteer. (H) Dot plot of paired geometric mean fluorescent intensity of FaO Blue in CD14+ gated PBMCs from SLE patient and respective age matched healthy volunteer (n=9). (I-M) Quantitative RT-PCR analysis of ISG15 (I) , CPT1A (J) , ACAT1 (K) , HADHA (L) and HADHB (M) in CD14+ myeloid cells isolated from PBMCs of SLE patients and respective age matched healthy volunteers. Data were normalized to 18 S rRNA. Paired t-test. (N) The heatmap shows expression profiling by array data from the GSE60359 dataset, comparing healthy controls and ISG15-deficient patients. Rows represent the genes of key FAO enzymes CRAT , ECHS1 , HADHA , ACAT1 , CPT1A , HADHB , PPARG , and PPARA , while columns represent six individual samples. Expression values were normalized using quantile normalization and scaled to z-scores. Colors indicate relative gene expression levels, as shown in the color bar. Asterisks represent - * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: Western Blot, Control, Gene Expression, Expressing, Biomarker Discovery, Isolation, Incubation, Staining, Flow Cytometry, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 is upregulated and facilitates fatty acid oxidation and mitochondrial activity in NB. a Western blot assay showing the p113 levels in normal dorsal ganglia (DG), tumor (T) tissues of NB, and cultured tumor cell lines. b Western blot assay indicating the expression of p113 and CUX1 isoforms (p200 and p110) in SH-SY5Y, SH-N-SH, BE(2)-C, and IMR32 cells stably transfected with empty vector (mock), ecircCUX1 , ecircCUX1 with ORF mutation ( ecircCUX1 Mut), scramble shRNA (sh-Scb), or sh-ecircCUX1. c Western blot assay showing the cytoplasmic and nuclear accumulation of p113 in SH-SY5Y cells stably transfected with mock or p113 . d Immunofluorescence assay revealing cytoplasmic and nuclear localization of p113 in SH-SY5Y and SK-N-SH cells stably transfected with Flag-tagged p113 (upper panel), and those in BE(2)-C and IMR32 cells (lower panel), with nuclei stained by DAPI (blue). Scale bar: 10 μm. e and f Heatmap (e) and quantification (f) of metabolite profiling assay indicating the fatty acid levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1 ( n = 3). g Seahorse extracellular flux assay showing the oxygen consumption rate (OCR) levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1, and those treated with BSA or oleic acid (OLE, 200 μmol·L − 1 , n = 4). h Relative NAD + /NADH ratio and ATP levels in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1, and those treated with BSA or OLE (200 μmol·L − 1 , n = 5). ANOVA compared the difference in f - h . * P < 0.05 vs. mock, sh-Scb, or sh-Scb + BSA. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in a - d , g and h

Article Snippet: Cells were seeded on coverslips and incubated with antibody specific for p113 (ABclonal Biotechnology Co., Ltd), Flag-tag (14793S, Cell Signaling Technology Inc.), 4-hydroxynonenal (4-HNE, MAB3249, Novus Biologicals, Ltd., Centennial, CO), or ALDH3A1 (ab186726, Abcam Inc.) at room temperature for 2 h. Then, coverslips were incubated with Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG, and stained with 4′,6-diamidino-2-phenylindole (DAPI, 300 nmol·L − 1 , Sigma).

Techniques: Activity Assay, Western Blot, Cell Culture, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, shRNA, Immunofluorescence, Staining, XF Assay

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 physically interacts with ZRF1 and BRD4 in NB cells. a Volcano plots showing differentially expressed genes (fold change> 1.5, P < 0.05) in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 . b Coomassie blue staining (left panel) and Venn diagram (right panel) revealing identification of p113-interacting proteins pulled down by p113 or Flag-tag antibody in SH-SY5Y cells stably transfected with 3Flag-tagged p113 , and those overlapped with transcription factors (TF) or epigenetic factors derived from ChIP-X and EpiFactors databases. c Co-IP and western blot assays indicating the interaction among p113, ZRF1, and BRD4 in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , scramble shRNA (sh-Scb), or sh-ecircCUX1. d Secondary co-IP assays showing protein interaction among p113, ZRF1, and BRD4 in SH-SY5Y cells stably transfected with HA-tagged p113 , Flag-tagged ZRF1 , and His-tagged BRD4 . e BiFC assay revealing the interaction of p113 with ZRF1 or BRD4 (arrowheads) in SH-SY5Y cells stably transfected with indicated constructs, with nuclei stained by DAPI. Scale bars: 10 μm. f and g Western blot assay (g) validating the knockdown of ZRF1 or BRD4 in SH-SY5Y cells stably transfected with scramble (Scb) or specific sgRNA for CRISPR interference (CRISPRi, f). Wild type (WT) cells were taken as negative controls. h Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with CRISPRi sgRNA specific against ZRF1 or BRD4 . i Schematic illustration of protein interaction among p113, ZRF1, and BRD4. j Dual-luciferase assay showing the activity of ZRF1 in NB cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1 ( n = 5). Fisher’s exact test for overlapping analysis in b . ANOVA compared the difference in j . * P < 0.05 vs. mock or sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c - e , g , h and j

Article Snippet: Cells were seeded on coverslips and incubated with antibody specific for p113 (ABclonal Biotechnology Co., Ltd), Flag-tag (14793S, Cell Signaling Technology Inc.), 4-hydroxynonenal (4-HNE, MAB3249, Novus Biologicals, Ltd., Centennial, CO), or ALDH3A1 (ab186726, Abcam Inc.) at room temperature for 2 h. Then, coverslips were incubated with Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG, and stained with 4′,6-diamidino-2-phenylindole (DAPI, 300 nmol·L − 1 , Sigma).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Staining, FLAG-tag, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, shRNA, Bimolecular Fluorescence Complementation Assay, Construct, CRISPR, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113/ZRF1/BRD4 complex promotes lipid metabolic reprogramming and mitochondrial complex I activity in NB cells. a Heatmap, distribution, and binding motif of ChIP-Seq (left panel) assay revealing genomic enrichment of ZRF1 in SH-SY5Y cells, while Venn diagram, heatmap, and GO pathway (right panel) showing identification of p113/ZRF1/BRD4 target genes by overlapping analysis of RNA-seq results upon p113 over-expression and ChIP-seq peaks of ZRF1 or BRD4. b ChIP-seq assay showing the binding peak of BRD4 or ZRF1 on promoter regions of ALDH3A1 , NDUFA1 , or NDUFAF5 in SH-SY5Y cells. c Western blot assay indicating the expression of ALDH3A1, NDUFA1, or NDUFAF5 in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi. d Schematic illustration showing the involvement of ALDH3A1, NDUFA1, or NDUFAF5 in lipid metabolic reprogramming and mitochondrial respiratory activity. e Relative OCR levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). f Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). ANOVA compared the difference in e and f . * P < 0.05 vs. mock+CRISPRi-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c , e and f

Article Snippet: Cells were seeded on coverslips and incubated with antibody specific for p113 (ABclonal Biotechnology Co., Ltd), Flag-tag (14793S, Cell Signaling Technology Inc.), 4-hydroxynonenal (4-HNE, MAB3249, Novus Biologicals, Ltd., Centennial, CO), or ALDH3A1 (ab186726, Abcam Inc.) at room temperature for 2 h. Then, coverslips were incubated with Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG, and stained with 4′,6-diamidino-2-phenylindole (DAPI, 300 nmol·L − 1 , Sigma).

Techniques: Activity Assay, Binding Assay, ChIP-sequencing, RNA Sequencing Assay, Over Expression, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 facilitates tumorigenesis and aggressiveness of NB cells via interacting with ZRF1. a and b Representative images (left panel) and quantification (right panel) of soft agar (a) and matrigel invasion (b) assays indicating the growth and invasion of NB cells stably transfected with empty vector (mock) or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 for CRISPRi. c Representative images (upper panel), in vivo growth curve, and weight at the end points (lower panel) of xenografts in nude mice formed by subcutaneous injection of SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 for CRISPRi ( n = 5 for each group). d Representative images (upper panel) and quantification (lower panel) of immunohistochemical staining showing expression of Ki-67 and CD31 within xenografts ( n = 5 for each group). Scale bars: 50 μm. e and f Representative in vivo images (e), hematoxylin & eosin staining (f, upper panel), lung metastatic counts (f, lower panel), and Kaplan–Meier curves (f, lower panel) of nude mice treated with tail vein injection of SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 for CRISPRi ( n = 5 for each group). Scale bars: 100 μm. ANOVA compared the difference in a - d and f . Log-rank test for survival comparison in f . * P < 0.05 vs. mock+CRISPRi-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in a and b

Article Snippet: Cells were seeded on coverslips and incubated with antibody specific for p113 (ABclonal Biotechnology Co., Ltd), Flag-tag (14793S, Cell Signaling Technology Inc.), 4-hydroxynonenal (4-HNE, MAB3249, Novus Biologicals, Ltd., Centennial, CO), or ALDH3A1 (ab186726, Abcam Inc.) at room temperature for 2 h. Then, coverslips were incubated with Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG, and stained with 4′,6-diamidino-2-phenylindole (DAPI, 300 nmol·L − 1 , Sigma).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, In Vivo, Injection, Immunohistochemical staining, Staining, Expressing

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: Therapeutic blocking p113-ZRF1 interaction inhibits NB progression. a 3D structure and sequences of inhibitory peptides (ZIP-12) for blocking interaction between p113 and ZRF1, and those of mutant control (Ctrl) peptides. b Confocal images showing the distribution of synthesized FITC-labeled Ctrl or ZIP-12 peptides (20 μmol·L − 1 , arrowheads) within cultured BE(2)-C cells, with nuclei and cellular membranes staining with DAPI or Dil. Scale bars: 10 μm. c Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. d Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. e In vivo images (left upper panel), growth curve (right panel), and weight at the end points (right panel) of xenografts formed by subcutaneous injection of BE(2)-C cells in nude mice ( n = 5 per group) that were treated with intravenous injection of Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (left lower panel). f In vivo imaging (left panel), lung metastatic colonization (right lower panel), and Kaplan–Meier curves (right lower panel) of nude mice ( n = 5 for each group) treated with tail vein injection of BE(2)-C cells, Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (right upper panel). Student’s t test or ANOVA compared the difference in d - f . Log-rank test for survival comparison in f . * P < 0.05, ** P < 0.01 vs. Ctrl. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in b - d

Article Snippet: Cells were seeded on coverslips and incubated with antibody specific for p113 (ABclonal Biotechnology Co., Ltd), Flag-tag (14793S, Cell Signaling Technology Inc.), 4-hydroxynonenal (4-HNE, MAB3249, Novus Biologicals, Ltd., Centennial, CO), or ALDH3A1 (ab186726, Abcam Inc.) at room temperature for 2 h. Then, coverslips were incubated with Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG, and stained with 4′,6-diamidino-2-phenylindole (DAPI, 300 nmol·L − 1 , Sigma).

Techniques: Blocking Assay, Mutagenesis, Synthesized, Labeling, Cell Culture, Staining, Co-Immunoprecipitation Assay, Western Blot, Activity Assay, In Vivo, Injection, In Vivo Imaging

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: CUX1 , ZRF1 , BRD4 and target genes are associated with poor outcome of NB patients. a Kaplan–Meier curves indicating overall survival of 498 well-defined NB cases (GSE62564) with high or low expression of CUX1 (cutoff value = 32.233), ZRF1 (cutoff value = 21.749), BRD4 (cutoff value = 652.58), ALDH3A1 (cutoff value = 1.181), NDUFA1 (cutoff value = 22.511), or NDUFAF5 (cutoff value = 7.964). b The positive expression correlation between ZRF1 and ALDH3A1 , NDUFA1 , or NDUFAF5 in 498 well-defined NB cases (GSE62564). c The mechanisms underlying p113-faciliated NB progression: as a novel protein encoded by ecircCUX1 , p113 cooperates with ZRF1 and BRD4 to activate the transcription of ALDH3A1 , NDUFA1 , or NDUFAF5 , resulting in promoted conversion of fatty aldehydes into fatty acids, fatty acid β-oxidation, mitochondrial complex I activity, growth, and aggressiveness of NB cells. Meanwhile, inhibitory peptides (ZIP-12) blocking p113-ZRF1 interaction suppresses tumor progression. Log-rank test for survival comparison in a . Pearson’s correlation coefficient for b

Article Snippet: Cells were seeded on coverslips and incubated with antibody specific for p113 (ABclonal Biotechnology Co., Ltd), Flag-tag (14793S, Cell Signaling Technology Inc.), 4-hydroxynonenal (4-HNE, MAB3249, Novus Biologicals, Ltd., Centennial, CO), or ALDH3A1 (ab186726, Abcam Inc.) at room temperature for 2 h. Then, coverslips were incubated with Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 488 goat anti-rabbit IgG, and stained with 4′,6-diamidino-2-phenylindole (DAPI, 300 nmol·L − 1 , Sigma).

Techniques: Expressing, Activity Assay, Blocking Assay