Journal: Nature Communications

Article Title: The invasion phenotypes of glioblastoma depend on plastic and reprogrammable cell states

doi: 10.1038/s41467-025-61999-1

Figure Lengend Snippet: A Multispectral IHC of U3054MG PDCX, with example staining of STEM121 in black, ANXA1 in brown, CD31 in red, MKI67 in yellow, and MBP in blue. Representative section from a total of n = 10 independent mouse replicates injected with U3054MG. B We segmented scans into 9 compartments (high-density tumor (1), medium-density tumor (2), low-density tumor (3), circle-shaped aggregates (4), tumor cells growing within close proximity to the vasculature (5), diffusely-growing elongated tumor cells in the corpus callosum (6), other diffusely invading cells (7), blood vessels (8), and mouse brain parenchyma (9). Created in BioRender. Nelander, S. (2025) https://BioRender.com/lpyogrt . C Scoring all PDCX models using 35 antibodies; upregulated and downregulated expression of proteins in named compartments for perivascular and diffusively invading cells. (Sections from n = 2 independent biological replicates (individual mice) were stained for each antibody, for each of the 6 cell lines). D Relative area of segmented compartments per PDCX cell line ( n = 3 independent biological replicates (individual mice). E Volcano plot indicating key differentially expressed proteins. The log10 p -values are obtained from a two-sided heteroscedastic t -test, not adjusted for multiple comparisons (Sections from n = 2 independent biological replicates (individual mice) were stained for each antibody, for each of the 6 cell lines). Source data are provided as a source data file.

Article Snippet: The sections were blocked with Normal Antibody Diluent (ImmunoLogic WellMed #UD09) for 30 min at room temperature, and primary antibodies STEM121 (1:500) (Takara #Y40410), ANXA1 (1:400) (CST, #32934S), RFX4 (1:500) (HPA #050527), and HOPX (1:1000) diluted in Normal Antibody Diluent were applied and incubated for 60 min at RT.

Techniques: Staining, Injection, Expressing

Journal: Nature Communications

Article Title: The invasion phenotypes of glioblastoma depend on plastic and reprogrammable cell states

doi: 10.1038/s41467-025-61999-1

Figure Lengend Snippet: A Human tissue microarray (TMA) staining of the tumor core, including patients U3013MG, U3180MG, and healthy brain tissue from HGCC. Staining includes CD31 in red, ANXA1 in brown, HOPX in blue, and RFX4 in cyan. The upper panel scale bar indicates 100 μm, while the lower panel scale bar is 20 μm. The stainings were repeated twice. Representative images chosen from n = 4 TMA cores from each patient. B Multivariate survival analysis using Cox regression on survival data from the HGCC, with age, sex, and transcriptional subtype as covariates, indicate associations between high ANXA1 protein (measured as the fraction of ANXA1-positive cells) and shorter survival, and between high RFX4 protein (measured as the fraction of RFX4-positive cells) and shorter survival. HR Hazard ratios, CI confidence intervals, and p -values are indicated in the figure. Two-sided test; no corrections for multiple comparisons were made. C Staining of the tumor core and D edge from three patients from BrainUK. Staining includes CD31 in red, ANXA1 in brown, HOPX in blue, RFX4 in cyan, and DAPI in black. The scale bar indicates 50 μm. The stainings were performed once. Representative images from n = 1 tumor sample section from each patient, and 7 neuropathologist-inspected fields per section. E ANXA1 and HOPX proteins are selectively found in perivascular and diffuse regions in BrainUK samples, as determined by a two-sided z -test for proportions, assessing differences in marker expression between perivascular and diffusely invading GBM cells. Asterisks indicate statistical significance: p = 0.0098 (*), p = 0.0012 (**). N = 14 patients, all listed in the table. No corrections were applied for multiple comparisons. (N/A means that this type of invasion was absent in the sample). Source data are provided as a source data file.

Article Snippet: The sections were blocked with Normal Antibody Diluent (ImmunoLogic WellMed #UD09) for 30 min at room temperature, and primary antibodies STEM121 (1:500) (Takara #Y40410), ANXA1 (1:400) (CST, #32934S), RFX4 (1:500) (HPA #050527), and HOPX (1:1000) diluted in Normal Antibody Diluent were applied and incubated for 60 min at RT.

Techniques: Microarray, Staining, Marker, Expressing

Journal: Nature Communications

Article Title: The invasion phenotypes of glioblastoma depend on plastic and reprogrammable cell states

doi: 10.1038/s41467-025-61999-1

Figure Lengend Snippet: A , B Mouse survival for ANXA1 -KO U3013MG ( n = 10 mice), HOPX -KO U3180MG ( n = 10), and RFX4 -KO U3180MG ( n = 10). C Automated segmentation into 8 compartments. Created in BioRender. Nelander, S. (2025) https://BioRender.com/lpyogrt . D – H Whole brain scans and staining for each genotype. n = 3 brains were analysed per group and the stainings were repeated four times. I – K Change in compartment area for each PDCX-KO compared to SCR control. In ( I , J ), N = 4 independent replicate mice were used, and in ( J ), N = 3 independent replicate mice were used. Each mouse is shown as a point. Error bars are 90% confidence intervals obtained from a two-sided t -test, based on independent mouse replicates. L Percentage of KI67+ cells in each genotype ( n = 4 independent biological replicates (individual mice) in each group were used in the ANXA1 knockout vs control comparison, and n = 3 independent biological replicates (individual mice), were used in the RFX4 and HOPX knockout vs control comparison. Points represent individual mice, the distribution represents all counted fields in all mice. * indicates two-sided t test, p = 0.0375, calculated for the mouse independent replicates). M Percentage of stellate cells in each genotype. ( n = 4 independent biological replicates, i.e., individual mice, in each group were used in the ANXA1 knockout vs control comparison, and n = 3 independent biological replicates, i.e., individual mice, were used in the RFX4 and HOPX knockout vs control comparison. Points represent individual mice, the distribution represents all counted fields in all mice; * indicates two-sided t test, p = 0.0139, calculated for the mouse independent replicates). Source data are provided as a source data file.

Article Snippet: The sections were blocked with Normal Antibody Diluent (ImmunoLogic WellMed #UD09) for 30 min at room temperature, and primary antibodies STEM121 (1:500) (Takara #Y40410), ANXA1 (1:400) (CST, #32934S), RFX4 (1:500) (HPA #050527), and HOPX (1:1000) diluted in Normal Antibody Diluent were applied and incubated for 60 min at RT.

Techniques: Staining, Control, Knock-Out, Comparison

Journal: Nature Communications

Article Title: The invasion phenotypes of glioblastoma depend on plastic and reprogrammable cell states

doi: 10.1038/s41467-025-61999-1

Figure Lengend Snippet: A – C Comparison of ANXA1 -KO U3013MG cells with wild-type shows a shift towards NPC-like and AC-like differentiation. (scRNAseq data from n = 2 pools of tumor cells isolated from PDCX brains, total of 12069 cells). D – F Shift of cell state distribution in RFX4 -KO U3180MG cells, towards an NPC-like, low-proliferating state. (scRNAseq data from n = 2 pools of cells isolated from PDCX brains, total of 10824 cells). G – I Shift of cell state distribution in HOPX -KO U3180MG cells towards MES-like state. (scRNAseq data from n = 2 pools of cells isolated from PDCX brains, total of 13270 cells).

Article Snippet: The sections were blocked with Normal Antibody Diluent (ImmunoLogic WellMed #UD09) for 30 min at room temperature, and primary antibodies STEM121 (1:500) (Takara #Y40410), ANXA1 (1:400) (CST, #32934S), RFX4 (1:500) (HPA #050527), and HOPX (1:1000) diluted in Normal Antibody Diluent were applied and incubated for 60 min at RT.

Techniques: Comparison, Isolation

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test