Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Cross-linking network of M2 and LAT1 (B) Log(RLU) normalised to siNT. Data are represented as mean ± SD (n = 3), with individual experiment means shown as circles. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. siSLC3A2 is replicated from . (C) Volcano plots showing fold-change (log2) versus significance (-log10 p-value) for SLC7A5 versus isotype control. Viral proteins are depicted in orange, SLC7A5 and SLC3A2 – in green. (D) Maximum intensity projection (MaxIP) of confocal images from A549 cells infected with WSN (Multiplicity of infection (MOI) 3) or mock at 12 hpi. Cells were stained for SLC7A5 (green), M2 (magenta), and nuclei (DAPI, cyan). Scale bars, 20 µm. (E) Pearson’s correlation coefficient quantification of SLC7A5 and M2 colocalisation in membrane regions. Analysis was performed in mock-infected cells (n = 64) and WSN-infected cells at 12 hpi (n = 119). Statistical significance was determined using the Mann-Whitney test (****p<0.0001). (F) Representative plane and intensity profile showing the co-localisation of SLC7A5 and M2 in A549 cells at 12 hpi.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Control, Infection, Staining, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Cross-linking network of M2 and LAT1 (B) Log(RLU) normalised to siNT. Data are represented as mean ± SD (n = 3), with individual experiment means shown as circles. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. siSLC3A2 is replicated from . (C) Volcano plots showing fold-change (log2) versus significance (-log10 p-value) for SLC7A5 versus isotype control. Viral proteins are depicted in orange, SLC7A5 and SLC3A2 – in green. (D) Maximum intensity projection (MaxIP) of confocal images from A549 cells infected with WSN (Multiplicity of infection (MOI) 3) or mock at 12 hpi. Cells were stained for SLC7A5 (green), M2 (magenta), and nuclei (DAPI, cyan). Scale bars, 20 µm. (E) Pearson’s correlation coefficient quantification of SLC7A5 and M2 colocalisation in membrane regions. Analysis was performed in mock-infected cells (n = 64) and WSN-infected cells at 12 hpi (n = 119). Statistical significance was determined using the Mann-Whitney test (****p<0.0001). (F) Representative plane and intensity profile showing the co-localisation of SLC7A5 and M2 in A549 cells at 12 hpi.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Control, Infection, Staining, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: Snapshot of in-cell protein contact sites reveals new host factors and hijacking of paraspeckles during influenza A virus infection

doi: 10.1101/2025.03.09.642134

Figure Lengend Snippet: (A) Cross-linking network of M2 and LAT1 (B) Log(RLU) normalised to siNT. Data are represented as mean ± SD (n = 3), with individual experiment means shown as circles. Statistical analysis was performed using ordinary one-way ANOVA. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. siSLC3A2 is replicated from . (C) Volcano plots showing fold-change (log2) versus significance (-log10 p-value) for SLC7A5 versus isotype control. Viral proteins are depicted in orange, SLC7A5 and SLC3A2 – in green. (D) Maximum intensity projection (MaxIP) of confocal images from A549 cells infected with WSN (Multiplicity of infection (MOI) 3) or mock at 12 hpi. Cells were stained for SLC7A5 (green), M2 (magenta), and nuclei (DAPI, cyan). Scale bars, 20 µm. (E) Pearson’s correlation coefficient quantification of SLC7A5 and M2 colocalisation in membrane regions. Analysis was performed in mock-infected cells (n = 64) and WSN-infected cells at 12 hpi (n = 119). Statistical significance was determined using the Mann-Whitney test (****p<0.0001). (F) Representative plane and intensity profile showing the co-localisation of SLC7A5 and M2 in A549 cells at 12 hpi.

Article Snippet: The following primary antibodies were used for western blot analysis: NONO mouse monoclonal (1:1,000, Proteintech, clone 2A2B10, catalogue no. 66361-1-Ig); NONO rabbit polyclonal (1:1,000, Proteintech, catalogue no. 11058-1-AP); SFPQ mouse monoclonal (1:1,000, Proteintech, clone 1G4A5, catalogue no. 67129-1-Ig); SFPQ rabbit polyclonal (1:1,000, Proteintech, catalogue no. 15585-1-AP); PSPC1 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 16714-1-AP); SLC7A5 rabbit polyclonal (1:1,000, Proteintech, catalogue no. 28670-1-AP); SLC7A5 mouse monoclonal (1:1,000, Proteintech, clone 2G5H3, catalogue no. 67951-1-Ig); SLC3A2 rabbit polyclonal (1:1,000, Proteintech, catalogue no 15193-1-AP); SLC3A2 mouse monoclonal (1:1,000, Proteintech, clone 2B10F5, catalogue no. 66883-1-Ig); NP mouse monoclonal (1:1,000, Abcam, clone C43, catalogue no. ab128193); M2 mouse (1:1,000, ThermoFisher, clone 14C2, catalogue no. MA1-082); PB1, PB2, M1 (1: 1000, Abcam ab22396), NS1 (1:1,000, ThermoFisher, MA5-35909); Vinculin mouse (1:5000, Merck, catalogue no. V9131); Cyclophilin B rabbit monoclonal (1:5,000, CST, clone D1V5J, catalogue no. 43603).

Techniques: Control, Infection, Staining, Membrane, MANN-WHITNEY

Journal: The FASEB Journal

Article Title: Targeting TUBG1 in RB1 ‐negative tumors

doi: 10.1096/fj.202403180RR

Figure Lengend Snippet: Influence of TUBG1, E2F1, and RB1 protein levels on the cytotoxic effect of L12 treatment. (A and B) The MCF10A cell lines used include: Control (MCF10A, non‐modified parental cells), MCF10A sh TUBG (stably expressing TUBG shRNA) and MCF10A sh TUBG TUBG1 (stably co‐expressing TUBG shRNA and a sh‐resistant TUBG1 gene). (A) MCF10A cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. DNA content was measured by nuclear counter to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction (indicative of dead cells). Histograms display the results, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (B) Western blotting (WB) was performed to analyze TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs illustrate relative protein expression (Student's t test, N = 3, * p < .05, ** p < .01). (C and D) The U2OS cell lines used include: Control (U2OS, non‐modified parental cells), U2OS sh E2F1 (transiently expressing E2F1 shRNA) and U2OS E2F1 sh E2F1 (transiently co‐expressing E2F1 sgRNA and a E2F1 gene). U2OS cells were treated with DMSO (vehicle) or 50 nM L12 for 24 h. (C) DNA content was measured to determine the cell cycle profile and the percentage of cells in the sub‐G1 fraction. Histograms show representative data, and graphs summarize the mean ± SD percentages of sub‐G1 cells ( N = 4; Student's t test, * p < .01, ** p < .01). (D) WB was used to analyze E2F1 and procaspase 3 protein levels in total lysates using anti‐E2F1 and anti‐procaspase 3 antibodies. GAPDH served as the loading control. Graphs display relative protein expression (Student's t test, N = 4, * p < .05, ** p < .01). The numbers above the blots (WB) represent the normalized intensity of the protein bands. (E and F) The A549 cell lines used include: Control (A549, non‐modified parental cells), A549 sg RB1 (stably expressing RB1 sgRNA) and A549 RB1 sgRNA RB1 (stably co‐expressing RB1 sgRNA and a sg‐resistant RB1 gene). (E) A549 cells were treated with DMSO (vehicle) or the indicated concentrations of L12 for 24 h. The cell cycle profile and percentage of sub‐G1 cells were determined. Histograms represent the results, and graphs show mean ± SD percentages of sub‐G1 cells ( N = 3; two‐way ANOVA, **** p < .0001). (F) WB analyzed TUBG and RB1 protein levels in total lysates using anti‐TUBG and anti‐RB1 antibodies. Actin served as the loading control. Graphs depict relative protein expression (Student's t test, N = 3, * p < .05, **** p < .0001). To ensure accurate comparisons of RB1 protein levels under different conditions, Western blot exposure times were optimized for each experiment to balance signal detection and prevent overexposure, enabling the detection of subtle differences in RB1 expression.

Article Snippet: Human TUBG1 single guide (sg; RRID:Addgene_104437) and short hairpin (sh) RNA (RRID:Addgene_87955), human sg‐resistant pcDNA3‐ TUBG1 (RRID:Addgene_104433), human E2F1 sh (RRID:Addgene_66883) and pcDNA3‐ TUBG2 (RRID:Addgene_171966) were prepared as previously reported., , , , The human pSG5L‐ RB1 and pcDNA‐ E2F1 constructs were kindly provided by Dr. W. Sellers (RRID:Addgene_10720 ) and Dr. J.R. Nevins (Duke University, USA ), respectively.

Techniques: Control, Modification, Stable Transfection, Expressing, shRNA, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Neuroprotective effects of ethanol extraction from Rubia yunnanensis Diels on chronic cerebral hypoperfusion: modulation of the System Xc-/GSH/GPX4 axis to alleviate oxidative stress and ferroptosis

doi: 10.3389/fphar.2025.1552228

Figure Lengend Snippet: Regulation of intracellular iron ion homeostasis and System Xc-/GSH/GHX4 pathway by RY-A. Expression of (A, B) TFRC, (A, C) FPN, (A, D) SLC7A11, (A, E) SLC3A2, (A, F) GSS, (A, G) GPX4 in rat hippocampal tissues was detected by Western blot. Data are expressed as mean ± SEM (n = 3). Compared to the Sham group, ****p < 0.0001; Compared to the 2VO group, ## p < 0.01, #### p < 0.0001. 2VO, 2-vessel occlusion; RY-A, Rubia yunnanensis alcohol extract.

Article Snippet: After that, the following antibodies were used: transferrin receptor antibody (1:5000; ab269513), anti-SLC7A11 antibody (1:1000; ab307601), anti-β-actin antibody (1:1000; ab8227), goat anti-rabbit IgG H&L (HRP) (1:10000; ab6721), and rabbit anti-mouse IgG H&L (HRP) (1:10000; ab6728), all from Abcam; anti-ferroportin/SLC40A1 antibody (1:500; 26601-1-AP) from Proteintech; and anti-CD98/SLC3A2 antibody (1:200; sc-136139), anti-GPX-4 antibody (1:100; sc-166120), and anti-GSS antibody (1:100; sc-365863) from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot