Journal: bioRxiv

Article Title: A stable subgenomic reporter coronavirus enables transcriptional profiling of bystander cells

doi: 10.64898/2026.02.27.708290

Figure Lengend Snippet: Reagents for analysing HCoV-OC43. A. Western blot of Mv.1.Lu cells infected with HCoV-OC43 at an MOI of 10 PFU/cell and harvested at the indicated time points, probed with two anti-Nucleocapsid (N) antibodies, derived from Sheep (Sh) polyclonal antiserum from the MRC PPU & CVR Coronavirus toolkit and a commercial Rabbit (Rb) polyclonal antiserum. Host HSP90 is included as a loading control. B . Immunofluorescence microscopy of HCoV-infected A549 cells, stained with Sheep anti-N and anti double-stranded RNA (dsRNA), a marker of RNA replication. C . Immunoprecipitation (IP) of lysates from HCoV-OC43-infected cells (MOI 3, 24 hpi) using Sheep anti-N and probed with Sheep anti-N and anti-Spike (S). Host HSP90 is included as a loading control. D . Immunofluorescence microscopy of HCoV-OC43-infected A549 cells, stained with mouse monoclonal (Mm) or rabbit polyclonal (Rb) anti-S. E . Silver stain (SS) and western blotting (WB) of virions purified by ultracentrifugation through a 30% sucrose cushion. The supernatant (sup) before and after sucrose cushion, and the pellet, containing purified virions, are shown. Viral structural proteins are indicated by red arrowheads and BSA from cell culture medium by a red asterisk. F . Immunofluorescence microscopy of HCoV-OC43-infected A549 cells stained with anti-SARS-CoV M. Scale bars represent 10 µm.

Article Snippet: Membranes were probed with sheep polyclonal anti-Nucleocapsid anti-serum (MRC PPU & CVR Coronavirus Toolkit, Sheep No. DA116, 1:1000) , rabbit anti-Nucleocapsid (40643-T62, Sino Biological, 1:1000), anti-Spike (PAB21478-100, The Native Antigen Company, 1:1000), anti-SARS-CoV Membrane (ABIN1887462, Antibodies Online, 1:200), anti-MHV nucleocapsid J3.3 ( ) (1:200), anti-GAPDH (AM4300, Invitrogen, 1:4000) and anti-HSP90 (MA5-35624, Invitrogen, 1:2000).

Techniques: Western Blot, Infection, Derivative Assay, Control, Immunofluorescence, Microscopy, Staining, Marker, Immunoprecipitation, Silver Staining, Purification, Cell Culture

Journal: Journal of Cancer

Article Title: C4orf3 Regulates HIF-1α Degradation Under Hypoxic Conditions and Contributes to the Malignant Phenotype in Small Cell Lung Cancer

doi: 10.7150/jca.127942

Figure Lengend Snippet: C4orf3 expression is upregulated under hypoxic conditions in SCLC cell lines. (A) Western blotting analysis showing increased C4orf3 protein levels after 0, 24, and 48 h of hypoxic culture. (B) Real-time PCR analysis demonstrating increased C4orf3 mRNA expression after 48 h of hypoxia compared with normoxia. *p < 0.05; error bars represent SD.

Article Snippet: Sections of 4 μm thickness were de-paraffinised, rehydrated, and subjected to immunohistochemical staining using primary antibodies against C4orf3 (No. 207622-T08; Sino Biological) and HIF-1α (No. ab51608; Abcam, Cambridge, UK).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of Cancer

Article Title: C4orf3 Regulates HIF-1α Degradation Under Hypoxic Conditions and Contributes to the Malignant Phenotype in Small Cell Lung Cancer

doi: 10.7150/jca.127942

Figure Lengend Snippet: C4orf3 promotes tumour growth in vitro and in vivo . (A) Proliferation of SCLC cell lines transfected with control or C4orf3 siRNA under normoxic (N) or hypoxic (H) conditions. (B) Subcutaneous xenograft model generated by implanting SBC-5 cells transfected with control or C4orf3 siRNA into the inguinal region of female nude mice (BALB/c nu/nu) (n = 4 per group). (C) Tumour volume measurements over time, collected at the end of the experiment. *p < 0.05; error bars represent SD.

Article Snippet: Sections of 4 μm thickness were de-paraffinised, rehydrated, and subjected to immunohistochemical staining using primary antibodies against C4orf3 (No. 207622-T08; Sino Biological) and HIF-1α (No. ab51608; Abcam, Cambridge, UK).

Techniques: In Vitro, In Vivo, Transfection, Control, Generated

Journal: Journal of Cancer

Article Title: C4orf3 Regulates HIF-1α Degradation Under Hypoxic Conditions and Contributes to the Malignant Phenotype in Small Cell Lung Cancer

doi: 10.7150/jca.127942

Figure Lengend Snippet: C4orf3 knockdown decreases HIF-1α expression in SCLC cells. (A) Immunofluorescence staining of SBC-5 cells showing perinuclear localisation of C4orf3 (green) and decreased HIF-1α (red) expression under hypoxia following C4orf3 knockdown. Nuclei were counterstained with DAPI (blue). (B) Western blotting analysis of C4orf3 and HIF-1α in SBC-5 cells cultured under hypoxic conditions with control or C4orf3 siRNA. (C) Western blotting analysis showing CoCl₂-induced upregulation of C4orf3 and HIF-1α under normoxia. (D) Western blotting analysis demonstrating attenuation of CoCl₂-induced HIF-1α expression following C4orf3 knockdown. (E) Positive correlation between C4orf3 and HIF-1α mRNA expression in SCLC cell lines based on the CellMiner-SCLC dataset.

Article Snippet: Sections of 4 μm thickness were de-paraffinised, rehydrated, and subjected to immunohistochemical staining using primary antibodies against C4orf3 (No. 207622-T08; Sino Biological) and HIF-1α (No. ab51608; Abcam, Cambridge, UK).

Techniques: Knockdown, Expressing, Immunofluorescence, Staining, Western Blot, Cell Culture, Control

Journal: Journal of Cancer

Article Title: C4orf3 Regulates HIF-1α Degradation Under Hypoxic Conditions and Contributes to the Malignant Phenotype in Small Cell Lung Cancer

doi: 10.7150/jca.127942

Figure Lengend Snippet: C4orf3 regulates HIF-1α stability through PIASy-mediated SUMOylation. (A) Negative correlation between C4orf3 and PIASy mRNA expression based on the CellMiner-SCLC dataset. (B) Western blotting analysis showing increased PIASy expression following C4orf3 knockdown in CoCl₂-treated SBC-5 cells. (C) Rescue of HIF-1α expression by simultaneous knockdown of PIASy in C4orf3-silenced SBC-5 cells under CoCl₂ treatment. (D) Immunoprecipitation analysis demonstrating increased SUMO1 binding to HIF-1α following C4orf3 knockdown.

Article Snippet: Sections of 4 μm thickness were de-paraffinised, rehydrated, and subjected to immunohistochemical staining using primary antibodies against C4orf3 (No. 207622-T08; Sino Biological) and HIF-1α (No. ab51608; Abcam, Cambridge, UK).

Techniques: Expressing, Western Blot, Knockdown, Immunoprecipitation, Binding Assay

Journal: Journal of Cancer

Article Title: C4orf3 Regulates HIF-1α Degradation Under Hypoxic Conditions and Contributes to the Malignant Phenotype in Small Cell Lung Cancer

doi: 10.7150/jca.127942

Figure Lengend Snippet: Immunohistochemical analysis of C4orf3 and HIF-1α expression in human SCLC specimens. Representative staining patterns from five clinical cases (×200 magnification).

Article Snippet: Sections of 4 μm thickness were de-paraffinised, rehydrated, and subjected to immunohistochemical staining using primary antibodies against C4orf3 (No. 207622-T08; Sino Biological) and HIF-1α (No. ab51608; Abcam, Cambridge, UK).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Journal of Cancer

Article Title: C4orf3 Regulates HIF-1α Degradation Under Hypoxic Conditions and Contributes to the Malignant Phenotype in Small Cell Lung Cancer

doi: 10.7150/jca.127942

Figure Lengend Snippet: C4orf3 contributes to malignant phenotypes of SCLC through HIF-1α. (A,B) Migration and invasion assays of SBC-5 cells treated with CoCl₂ following transfection with control or C4orf3 siRNA. Nuclei were counterstained with DAPI (blue). *p < 0.05; error bars represent SD. (C,D) Western blotting analysis showing hypoxia- or CoCl₂-induced upregulation of EMT-related markers (SLUG, SNAI1, and TWIST) and attenuation of their expression following C4orf3 knockdown. (E) Gene set enrichment analysis of SBC-5 cells cultured under hypoxic conditions for 48 h, with or without C4orf3 knockdown. (F) Proliferation assay of SBC-5 cells cultured in CoCl₂ showing reduced growth following C4orf3 knockdown. (G) Apoptosis assay showing an increased apoptotic fraction after C4orf3 silencing following CoCl₂ treatment.

Article Snippet: Sections of 4 μm thickness were de-paraffinised, rehydrated, and subjected to immunohistochemical staining using primary antibodies against C4orf3 (No. 207622-T08; Sino Biological) and HIF-1α (No. ab51608; Abcam, Cambridge, UK).

Techniques: Migration, Transfection, Control, Western Blot, Expressing, Knockdown, Cell Culture, Proliferation Assay, Apoptosis Assay

Journal: Journal of Cancer

Article Title: C4orf3 Regulates HIF-1α Degradation Under Hypoxic Conditions and Contributes to the Malignant Phenotype in Small Cell Lung Cancer

doi: 10.7150/jca.127942

Figure Lengend Snippet: Proposed model of C4orf3-mediated regulation of HIF-1α in SCLC. Under hypoxic conditions, C4orf3 suppresses PIASy-mediated SUMOylation, thereby stabilising HIF-1α and promoting malignant phenotypes such as proliferation, migration, and invasion in SCLC cells.

Article Snippet: Sections of 4 μm thickness were de-paraffinised, rehydrated, and subjected to immunohistochemical staining using primary antibodies against C4orf3 (No. 207622-T08; Sino Biological) and HIF-1α (No. ab51608; Abcam, Cambridge, UK).

Techniques: Migration