Journal: bioRxiv

Article Title: RSV Infects the Human Nasal Epithelium via the Basolateral Route with Distinct Subgroup Infectivity and Basal Cell Tropism

doi: 10.64898/2026.03.05.709813

Figure Lengend Snippet: (A) Apically released plaque forming units of RSV/B/BA from each transwell replicate of one adult HNO-ALI line at 5 and 8 dpi. LOD = limit of detection. (B) Distribution of cell populations from basolateral mock and RSV/B/BA inoculation at 5 and 8 dpi. (C) Representative spectral flow cytometry plots showing gating strategy to identify Krt5+, Krt23+, and Ace-tub+ cell populations in basolateral mock and RSV/B/BA inoculated HNO-ALIs at 5 dpi. Further gating of RSV F protein was used to determine RSV infection in each cell population. (D) Pie charts summarizing the average proportion of each cell population in basolateral mock and RSV/B/BA inoculated HNO-ALIs at 5 dpi. (E) Pie charts summarizing RSV/B/BA infection in each cell population at 5 dpi.

Article Snippet: Immunostaining for intracellular proteins was performed using the following primary conjugated antibodies: Krt5 (1:5000, Abcam, Catalog number: ab224985), Krt17 (1:100, Abcam, Catalog number: ab185032), Krt23 (1:100, Novus Biologicals, Catalog number: NBP2–22590AF488), acetylated α-tubulin (1:100, Santa Cruz, Catalog number: sc-23950 AF594), and RSV-F protein (1:100, Novus Biologicals, Catalog number: NB100–64495JF646).

Techniques: Flow Cytometry, Infection

Journal: Frontiers in Oncology

Article Title: cGAS/STING-mediated upregulation of NKG2D ligands in LSCs contributes to enhanced sensitivity to NK cells

doi: 10.3389/fonc.2026.1764427

Figure Lengend Snippet: PARP1 is involved in the regulation of NKG2D ligand expression in LSCs. (A) WB was used to detect the knockdown efficiency of PRAP1 in LSCs. GAPDH was employed as an internal loading control. (B) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B in PARP1-stable knockdown or -scramble control LSCs were detected by flow cytometry (BD FACSCalibur). Mouse IgG-APC was used as isotype control. (C) Formation of γH2AX foci mediated by the pretreatment of 1 μM PARP1 inhibitor (PARPi) for 6 hours in LSCs was detected by immunofluorescence and observed in a fluorescence microscope (Zeiss). DMSO was used as vehicle control. Scale bars, 10 μm. (D) The protein expression level of γH2AX, H2AX, phosphorylated-ATM (Ser1981), ATM, and PARP1 in LSCs pretreated with 1μM PARPi or Vehicle for 6 hours were detected by WB. GAPDH was employed as an internal loading control. (E) Quantification of the WB shown in (D) . The Integrated Optical Density of bands was measured by the software ImageJ. Data was presented as mean ± SD from three experiments. (F) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B on the cell surface of LSCs treated with 1 μM PARPi or co-treated with 1 μM PARPi and 1 μM ATM inhibitor (ATMi) for 6 hours were measured by flow cytometry (BD FACSCalibur). DMSO was used as Vehicle control. Mouse IgG-APC was used as isotype control. ***p < 0.001. ns, no significance.

Article Snippet: Antibodies against PARP1 (Cat #: 66520-1-Ig, Proteintech), cGAS (Cat # : 79978, Cell signaling technology), STING (Ca # : 13647, Cell signaling technology), phospho-STING (Ser366) (Cat#:72650, Cell signaling technology), phospho-TBK1 (Ser172) (Cat#: 5483, Cell signaling technology), TBK1 (Cat#: 3504, Cell signaling technology), IRF3(Cat#: 4302, Cell signaling technology), phospho-IRF3 (Ser396) (Cat#: 4947, Cell signaling technology), ATM (Cat#: 27156-1-AP, Proteintech), phospho-ATM (Ser1981) (Cat#: 5883, Cell signaling technology), phospho-γH2AX (Ser139) (Cat#: 83307-2-RR, Proteintech), H2AX (Cat#: 39689, Proteintech), GAPDH (Cat#: 60004-1-Ig, Proteintech), α-Tubulin (Cat#: 14555-1-AP, Proteintech) were used as primary antibodies in the western blotting assay; Goat Anti-Mouse IgG antibody, HRP conjugated (Cat#: 2900264, Millipore), and Goat Anti-Rabbit IgG antibody, HRP conjugated (Cat#: 3256751, Millipore) were used as second antibodies.

Techniques: Expressing, Knockdown, Control, Flow Cytometry, Immunofluorescence, Fluorescence, Microscopy, Software

Journal: Nature Communications

Article Title: Targeting LRBA triggers CTLA4 degradation and antitumor immunity for cancer immunotherapy

doi: 10.1038/s41467-025-67365-5

Figure Lengend Snippet: A Representative image of LRBA (top) and CTLA4 (bottom) IHC staining in tumor tissues of colorectal cancer patients ( n = 94 patients). B Correlation between LRBA and CTLA4 expression in tumor tissue (left, n = 94 patients) and para-tumor tissue (right, n = 94 patients) of colorectal cancer patients. Data are pooled from the normalized number of CTLA4 + cells and LRBA + cells analyzed in A. C Tumor volume of MC38 tumor-bearing WT ( n = 10 mice) or LRBA -/- ( n = 8 mice) mice and performed once. D Chemical structure of LC427. E, F Relative activity of LC427 in HEK293T cells transfected with Sm-LRBA PH and WT CTLA4 CT-Lg ( E , n = 4 biological replicates) or mutant CTLA4 CT-Lg (Y201V) ( F , n = 4 biological replicates). G The effect of LC427 on the interaction between LRBA and WT CTLA4 in HEK293T cells transfected with LRBA PH and WT CTLA4. Co-immunoprecipitation (Co-IP) was performed using an anti-Flag antibody bead, followed by immunoblotting with the indicated antibodies. The relative density values for each band beneath the corresponding Western Blot lanes and from two independent experiments (mean). Molecular weight markers (kDa) on the right of the relevant Western Blot images. H The effect of LC427 on the interaction between LRBA and CTLA4 in human PBMCs. The relative density values for each band beneath the corresponding Western Blot lanes and from two independent experiments (mean). Molecular weight markers (kDa) on the right of the relevant Western Blot images. I The effect of LC427 on the interaction between purified Sm-LRBA PH and WT CTLA4 CT-Lg ( n = 4 biological replicates). J ITC binding curves for titration of LC427 (120 µM) into LRBA (10 µM). The upper part shows the thermogram after baseline correction and the bottom part shows the binding isotherm. K Crystal structure of LRBA (PDB code 1T77) in complex with LC427. Hydrogen bonds with Asn2349 and Arg2345 (red dashed lines), hydrophobic bonds with Ala2313 and Phe2340 (green dashed lines), and π-π stacking interaction with Trp2314 are shown. L Relative activity of LC427 in HEK293T cells transfected with mutant Sm-LRBA PH (N2349D) and WT CTLA4 CT-Lg ( n = 4 biological replicates). M The effect of LC427 on the interaction of WT CTLA4 with WT LRBA-PH or mutant LRBA-PH (N2349D) in HEK293T cells. The relative density values for each band beneath the corresponding Western Blot lanes and from two independent experiments (mean). Molecular weight markers (kDa) on the right of the relevant Western Blot images. N LC427 disrupts the interaction of intracellular CTLA4 with LRBA. As diagrammed on the right, we differentiated between surface CTLA4 bound to anti-CTLA4 mAbs and intracellular CTLA4 not bound to the antibody. Immunoprecipitates and total cell lysates (input) were probed with either anti-CTLA4 or anti-LRBA (left). The relative density values for each band beneath the corresponding Western Blot lanes and from two independent experiments (mean). Molecular weight markers (kDa) on the right of the relevant Western Blot images. Data are presented as mean ± SEM. Each biochemical experiment was at least repeated twice independently. The n number represents biological replicates (individual mice) obtained from one independent experiment. Significance was assessed using two-way ANOVA, Kaplan-Meier survival analyses or Spearman correlation coefficient. Source data are provided as a Source Data file.

Article Snippet: Antibodies (Supplementary Table ) were diluted as follows: MYC tag antibody (1:2000, 16286-1-AP, ProteinTech), DYKDDDDK tag antibody (1:5000, 80010-1-RR,ProteinTech), Anti-LRBA (1:2000, ab191174, Abcam), Anti-CTLA4 (F-8) (1:2000, sc-376016, Santa Cruz Biotechnology), Bcl-2 antibody (1:1000, 15071, CST), Beta-Actin (1:2000, 81115-1-RR, ProteinTech), NFκB1 antibody (1:2000, 14220-1-AP, ProteinTech), RELA antibody (1:2000, 14220-1-AP, ProteinTech), BCL2 antibody (1:2000, 26593-1-AP, ProteinTech), Caspase3 antibody (1:2000, 19677-1-AP, ProteinTech), AP1 antibody (1:2000, 24909-1-AP, ProteinTech), STAT2 antibody (1:2000, 16674-1-AP, ProteinTech), AKT antibody (1:2000, 10176-2-AP, ProteinTech), STAT5 antibody (1:2000, 13179-1-AP, ProteinTech).

Techniques: Immunohistochemistry, Expressing, Activity Assay, Transfection, Mutagenesis, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Molecular Weight, Purification, Binding Assay, Titration

Journal: Nature Communications

Article Title: Targeting LRBA triggers CTLA4 degradation and antitumor immunity for cancer immunotherapy

doi: 10.1038/s41467-025-67365-5

Figure Lengend Snippet: A, B LC427 increases CTLA4 degradation in HEK293T cells with CTLA4 and LRBA PH over-expression in a time-dependent manner ( A ) and in a dose-dependent manner ( B ). The relative density values for each band beneath the corresponding Western Blot lanes and from two independent experiments (mean). Molecular weight markers (kDa) on the right of the relevant Western Blot images. C Inhibitor of lysosomal degradation reverses the effect of LC427 on CTLA4 degradation in HEK293T cells with CTLA4 and LRBA PH over-expression (CHX, Cycloheximide; CQ, Chloroquine). The relative density values for each band beneath the corresponding Western Blot lanes and from two independent experiments (mean). Molecular weight markers (kDa) on the right of the relevant Western Blot images. D Confocal microscopy shows that LC427 reduces CTLA4 expression and localization in the lysosome (CQ, Chloroquine). E The quantification of YFP-CTLA4 intensity in YFP-CTLA4 positive cells and LysoTracker localization to YFP-CTLA4 positive structures in ( D ) ( n = 6 biological replicates). F The quantification of intracellular CTLA4 + (top) and surface CTLA4 + (bottom) in mouse naïve or activated CD8 + T cells, CD4 + T cells, and Foxp3 + Treg cells with the indicated concentration of LC427 (0, 5, 10, 20 µM) treatment for 5 days ( n = 3 biological replicates). G The quantification of surface CTLA4 + in human naïve or activated CD8 + T cells (left) and CD4 + T cells (right) with indicated LC427 treatment for 5 days ( n = 3 biological replicates). H, I The effects of LC427 on proliferation ( H , n = 5 biological replicates) and apoptosis ( I , n = 3 biological replicates) in mouse CD4 + T cells and CD8 + T cells after 5-day treatment. J The effects of LC427 on apoptosis in human CD8 + T cells (left) and CD4 + T cells (right) after 5-day treatment ( n = 3 biological replicates). K The quantification of absolute alive cell number of human T cells after LC427 treatment for 5 days ( n = 3 biological replicates). L The effects of LC427 on IL-2 (top) and IFN-γ (bottom) expression in human peripheral blood mononuclear cells (hPBMCs) after 3-day treatment ( n = 3 biological replicates). M The effect of WT OT1 CD8 + T cells pretreated by LC427, anti-PD1 antibody, or anti-CTLA4 antibody on cell viability of MC38-OVA tumor ( n = 3 biological replicates). N The effect of Lrba-Knockdown ( shLrba ) or Control-Knockdown ( shCtrl ) OT1 CD8 + T cells pretreated by LC427 on cell viability of MC38-OVA tumor ( n = 3 biological replicates). The relative density values for each band beneath the corresponding Western Blot lanes. Molecular weight markers (kDa) on the right of the relevant Western Blot images. Data are mean ± SEM. Each biochemical experiment was at least repeated twice independently. Statistical significance was assessed using one-way ANOVA. ns, Source data are provided as a Source Data file.

Article Snippet: Antibodies (Supplementary Table ) were diluted as follows: MYC tag antibody (1:2000, 16286-1-AP, ProteinTech), DYKDDDDK tag antibody (1:5000, 80010-1-RR,ProteinTech), Anti-LRBA (1:2000, ab191174, Abcam), Anti-CTLA4 (F-8) (1:2000, sc-376016, Santa Cruz Biotechnology), Bcl-2 antibody (1:1000, 15071, CST), Beta-Actin (1:2000, 81115-1-RR, ProteinTech), NFκB1 antibody (1:2000, 14220-1-AP, ProteinTech), RELA antibody (1:2000, 14220-1-AP, ProteinTech), BCL2 antibody (1:2000, 26593-1-AP, ProteinTech), Caspase3 antibody (1:2000, 19677-1-AP, ProteinTech), AP1 antibody (1:2000, 24909-1-AP, ProteinTech), STAT2 antibody (1:2000, 16674-1-AP, ProteinTech), AKT antibody (1:2000, 10176-2-AP, ProteinTech), STAT5 antibody (1:2000, 13179-1-AP, ProteinTech).

Techniques: Over Expression, Western Blot, Molecular Weight, Confocal Microscopy, Expressing, Concentration Assay, Knockdown, Control

Journal: Nature Communications

Article Title: Targeting LRBA triggers CTLA4 degradation and antitumor immunity for cancer immunotherapy

doi: 10.1038/s41467-025-67365-5

Figure Lengend Snippet: A, B Normalized body weight ( A , n = 10 mice) and survival ( B , n = 10 mice) curves of mice treated with 3% DSS and receiving LC427 or anti-CTLA4 mAb. C, D Representative H&E staining ( C , n = 5 mice) and the histological scores ( D , n = 5 mice) of the colon from mice treated with 3% DSS and receiving LC427 or anti-CTLA4 mAb. E Concentrations of CXCL1, TNF-α, IL-6, and IFN-γ in the serum of mice treated with 3% DSS and receiving LC427 or anti-CTLA4 ( n = 5 mice). F Quantification of CD11b, CD68, CD163, and Ly6G IHC staining in the colon from mice with 3% DSS and receiving LC427 or anti-CTLA4 ( n = 6 mice). Data are mean ± SEM. The n number represents biological replicates (individual mice) obtained from one independent experiment. Statistical significance was assessed using Kaplan-Meier survival analyses and one-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: Antibodies (Supplementary Table ) were diluted as follows: MYC tag antibody (1:2000, 16286-1-AP, ProteinTech), DYKDDDDK tag antibody (1:5000, 80010-1-RR,ProteinTech), Anti-LRBA (1:2000, ab191174, Abcam), Anti-CTLA4 (F-8) (1:2000, sc-376016, Santa Cruz Biotechnology), Bcl-2 antibody (1:1000, 15071, CST), Beta-Actin (1:2000, 81115-1-RR, ProteinTech), NFκB1 antibody (1:2000, 14220-1-AP, ProteinTech), RELA antibody (1:2000, 14220-1-AP, ProteinTech), BCL2 antibody (1:2000, 26593-1-AP, ProteinTech), Caspase3 antibody (1:2000, 19677-1-AP, ProteinTech), AP1 antibody (1:2000, 24909-1-AP, ProteinTech), STAT2 antibody (1:2000, 16674-1-AP, ProteinTech), AKT antibody (1:2000, 10176-2-AP, ProteinTech), STAT5 antibody (1:2000, 13179-1-AP, ProteinTech).

Techniques: Staining, Immunohistochemistry