Journal: bioRxiv

Article Title: Modulation of β-Catenin is important to promote WNT expression in macrophages and mitigate intestinal injury

doi: 10.1101/2024.09.21.614209

Figure Lengend Snippet: (A) TOPflash assay to determine WNT activity in BMMɸ CM. CM from LiCl-treated BMMɸ showed lower WNT activity compared to untreated BMMɸ CM to. (B) qPCR analysis of RNA from BMMΦ treated with LiCl demonstrated downregulation of Wnt mRNA expression in comparison to the untreated BMMɸ. (C) TOPflash assay using CM from BMMɸ treated with Tankyrase inhibitor XAV939 demonstrated higher WNT activity compared to untreated BMMɸ CM. PBS and LiCl were used as negative and positive control, respectively. (D) qPCR analysis of RNA from XAV939 treated BMMΦ showed significantly higher Wnt expression compared to untreated BMMɸ. (E) ELISA to detect secretory WNTs in BMMɸ conditioned media. (F) TOPflash assay demonstrated higher WNT activity in CM from h-Mɸ treated with XAV939 compared to untreated h-Mɸ CM. (G) qPCR analysis of RNA from XAV939 treated h-Mɸ shows the significantly higher WNT expression compared to untreated h-Mɸ. (H) Schematic representation of canonical WNT signaling with/without pharmacological inhibition of β-Catenin. Data presented as the mean ± SD. (Significant level, *: p<0.05, **: p<0.005, ***: p<0.0005, ****: p<0.00005, NS: Not significant).

Article Snippet: After protein transfer, the membrane was blocked with 5% skim milk for one hour followed by incubation overnight at 40C with primary antibody against β-Catenin (1:1000 dilution) (Cell-Signaling, Cat no. 9582) and TCF-4 (Cell Signaling, Cat no:2569T).

Techniques: TOPFlash assay, Activity Assay, Expressing, Comparison, Positive Control, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: bioRxiv

Article Title: Modulation of β-Catenin is important to promote WNT expression in macrophages and mitigate intestinal injury

doi: 10.1101/2024.09.21.614209

Figure Lengend Snippet: (A) Schematic representation of β-Catenin ChIP followed by DNA sequencing in C57BL/6 mouse BMMɸ. (B) ChIP sequence data showed the presence of multiple WNT promoters. WNT9b and WNT5a promoters are top in the list based on confidence score. (C) Construct map of a luciferase reporter system under WNT5a or WNT9b promoter. (D) Luciferase reporter assay to determine WNT5a or WNT9b promoter activity. HEK293T cells transfected with scramble or pTA-WNT 5a or pTA-WNT 9b promoter plasmid followed by detection of luciferase activity. Transfected HEK293T cells treated with XAV939 demonstrated significant increase in luciferase activity (pTA-WNT 5a promoter p<0.00005) and (pTA-WNT 9b promoter p<0.005) compared to scramble plasmid transfected H293T cells. (E) qPCR analysis of β-Catenin CHIP sample from transfected HEK293T cells showed increase in presence of WNT5a promoter 2.35-fold (p<0.005) and WNT9b promoter 20.36-fold (p<0.005) in comparison with their respective IgG control. Data presented as the mean ± SD. (Significant level, **: p<0.005, ****: p<0.00005).

Article Snippet: After protein transfer, the membrane was blocked with 5% skim milk for one hour followed by incubation overnight at 40C with primary antibody against β-Catenin (1:1000 dilution) (Cell-Signaling, Cat no. 9582) and TCF-4 (Cell Signaling, Cat no:2569T).

Techniques: DNA Sequencing, Sequencing, Construct, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Comparison, Control

Journal: bioRxiv

Article Title: Modulation of β-Catenin is important to promote WNT expression in macrophages and mitigate intestinal injury

doi: 10.1101/2024.09.21.614209

Figure Lengend Snippet: (A) Schematic representation of experimental plan. (B) Body weight of mice at post irradiation time points (n=15 mice per group). (C) Kaplan–Meier survival (Mantel-Cox test) analysis of C57BL/6 mice exposed to WBI (10Gy) shows 80% survival (p<0.005) after pre-conditioned BMMɸ treatment compared to 40% survival (p<0.05) in mice receiving untreated BMMɸ. (D) Hematoxylin and Eosin staining of jejunum section from mice (upper panel 2x, lower panel 10x magnification). (E-F) Histogram showing villus length and crypt depth in jejunal section of C57BL/6 mice. Pre-conditioned BMMɸ treatment showing less reduction of villi length compared to irradiated mice receiving native BMMɸ or no cell therapy. (G) Ki-67 immunostaining of jejunal section (upper panel 2x, lower panel 10x magnification). (H) Histogram demonstrating higher number of Ki67+ cells per intestinal crypt in irradiated mice treated with pre-conditioned BMMɸ. (I) Histogram demonstrating significant low serum dextran level in mice treated with pre-conditioned BMMɸ compared to irradiated group (n=5 mice per group). (J) Representative microscopic images (x40 magnification) of jejunal sections immune-stained with anti β-Catenin antibody to determine β-Catenin nuclear localization. Nucleus stained with hematoxylin. Pre-conditioned BMMɸ treatment showed more nuclear β-Catenin staining (dark brown; indicated with arrows), compared to mice receiving native BMMɸ or no cell therapy. (K) Nuclear β-Catenin count: each data point is the average of the number of β-Catenin-positive nucleus from 15 crypts per field, 5 fields per mice. Number of β-Catenin-positive nucleus in irradiated C57BL/6 mice receiving pre-conditioned BMMɸ is higher compared to mice receiving native BMMɸ or no cell therapy. Treatment with native BMMɸ or without cell therapy following irradiation showed significantly fewer β-Catenin-positive nuclei than the non-irradiated control. Data presented as the mean ± SD. (Significant level, **: p<0.0005, ***: p<0.0005)

Article Snippet: After protein transfer, the membrane was blocked with 5% skim milk for one hour followed by incubation overnight at 40C with primary antibody against β-Catenin (1:1000 dilution) (Cell-Signaling, Cat no. 9582) and TCF-4 (Cell Signaling, Cat no:2569T).

Techniques: Irradiation, Staining, Immunostaining, Control

Journal: bioRxiv

Article Title: Modulation of β-Catenin is important to promote WNT expression in macrophages and mitigate intestinal injury

doi: 10.1101/2024.09.21.614209

Figure Lengend Snippet: (A) Schematic representation of PBI and EV treatment. (B) Kaplan–Meier survival (Mantel-Cox test) analysis of C57BL/6 mice receiving pre-conditioned or native BMMɸ derived EVs (200µg) at 24hrs and 48hrs post PBI (12 Gy). Mice receiving pre-conditioned BMMɸ derived EVs showed 80% survival compared to mice receiving native BMMɸ EVs (p<0.05; Log-rank (Mantel–Cox) test) or untreated mice (p<0.0005; Log-rank (Mantel–Cox) test) (n=10 per group). (C) The body weight analysis showed improvement with EVs treatment. (D) Schematic representation of WBI and EV treatment. (E) The body weight analysis showed improvement with EV treatment. (F) Kaplan–Meier survival (Mantel-Cox test) analysis of C57BL/6 mice exposed to WBI (9Gy) shows significant (80%) (p<0.0005) improvement in survival after treatment with pre-conditioned BMMɸ EVs. (n=10 mice per group) compared to mice receiving native BMMɸ EVs (p<0.05; Log-rank (Mantel–Cox) test) or untreated mice (p<0.0005; Log-rank (Mantel–Cox) test) (n=10 per group). (G) Histogram demonstrating significantly low serum dextran level in irradiated mice treated with pre-conditioned BMMɸ EVs compared to irradiated control (n=5 mice per group). (H) qPCR analysis of β-Catenin target genes from C57BL6 mice crypt epithelial cells. (I) Confocal microscopic images of crypt organoids from Lgr5-EGFP-CRE-ERT2; R26-ACTB-tdTomato-EGFP mice. Pre-conditioned BMMɸ EVs rescued the LGR5 +ve (GFP +ve cells) ISCs from radiation injury. Histogram demonstrating the improvement in irradiated organoid growth (J) and ATP uptake (K) with pre-conditioned BMMɸ EVs treatment compared to untreated irradiated control. (L) Microscopic images of organoids from human intestinal surgical specimens. (M) Histogram demonstrating the higher budding organoid ratio in EVs treated group compared to irradiated control. (N) Histogram demonstrating significant increase in ATP uptake with pre-conditioned BMMɸ derived EVs treatment compared to untreated irradiated control. (O) Schematic representation of the treatment plan for lineage tracing assay. (P) Confocal microscopic images of the jejunum section from Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice. tdTomato (tdT)-positive cells and Lgr5+ GFP+ cells are shown in red and green respectively. Nuclei are stained with DAPI (blue). (Q) Histogram representing the number of tdT-positive cells containing villi. Pre-conditioned BMMɸ EVs treatment increase in red cell containing villi (n=3 mice per group) compared to native BMMɸ EVs treatment or irradiated control. Data presented as the mean ± SD. (Significant level, *: p<0.05, **: p<0.005, ***: p<0.0005, ****: p<0.0005).

Article Snippet: After protein transfer, the membrane was blocked with 5% skim milk for one hour followed by incubation overnight at 40C with primary antibody against β-Catenin (1:1000 dilution) (Cell-Signaling, Cat no. 9582) and TCF-4 (Cell Signaling, Cat no:2569T).

Techniques: Derivative Assay, Irradiation, Control, Staining

Journal: bioRxiv

Article Title: Postnatal Tendon Growth and Remodeling Requires Platelet-Derived Growth Factor Receptor Signaling

doi: 10.1101/208991

Figure Lengend Snippet: (A) Representative immunoblots of serum-starved tendon fibroblasts treated with 20 ng/ml of PDGF-BB for 30 minutes with or without 1 μM of the PDGFR inhibitor CP-673,451. A Coomassie stained membrane is shown as a loading control. (B) Representative immunoblots of 3-day overloaded plantaris tendons demonstrating the ability of CP-673,451, to inhibit phosphorylation of PDGFRα and PDGFRβ in vivo. Phosphorylation of Akt and ERK1/2 were also inhibited by PDGFR inhibitor treatment. β-tubulin is shown as a loading control. (C) Immunohistochemistry of 3-day overloaded plantaris tendons treated with vehicle or PDGFR inhibitor showing a decrease in the abundance of p-PDGFRα/PDGFRβ-expressing cells in the overloaded plantaris tendons treated with CP-673,451 relative to vehicle-treated controls. Scale bars are 10 μm. DAPI, blue; WGA, green; p-PDGFRα Y849 /PDGFRβ Y857 , red.

Article Snippet: Primary rabbit antibodies against p-PDGFRα Y849 /PDGFRβ Y857 (1:100; #3170, Cell Signaling Technology, Danvers, MA, USA) were also used on slides from C57BL/6 mice to determine differences in the level of PDGFR phosphorylation in response to vehicle or PDGFR inhibitor treatment.

Techniques: Western Blot, Staining, In Vivo, Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Transient Overexpression of VEGF-A in Adipose Tissue Promotes Energy Expenditure via Activation of the Sympathetic Nervous System

doi: 10.1101/323659

Figure Lengend Snippet: (A) Q-PCR analysis of β-oxidation-related genes, namely Acadl, Acadm, Acads, Cptla, Cpt2 , and Echsl in sWAT of VEGF Tg mice and their littermate controls after HFD plus Dox feeding for 7 days (n = 5 per group, Student’s t-test , *, p < 0.05). (B) Q-PCR analysis of mitochondrial biogenetic genes including Pgcla, Nrf1, Cox IV , and Tfam in sWAT of VEGF Tg mice and their littermate controls after HFD plus Dox feeding for 7 days (n = 5 per group, Student’s t-test , *, p < 0.05). (C) The mitochondrial DNA (mtDNA) content in sWAT of VEGF Tg mice and their littermate controls after HFD plus Dox feeding for 7 days. The copy number of mtDNA was calculated by the ratio of the mtDNA gene NADH dehydrogenase alpha 1 ( Nadha1 ) to the nuclear gene lipoprotein lipase ( Lpl ) (n = 5 per group, Student’s t-test , **, p < 0.01). (D) Immunofluorescent (IF) staining with α-COX IV antibody (green) in sWAT of VEGF Tg mice and their littermate controls after HFD plus Dox feeding for 7 days. The nuclei were stained with DAPI (blue) (Scale bar, 20 μm). (E) Western blotting analysis of protein levels of SDHA with α-SDHA antibody in sWAT of VEGF Tg mice and their littermate controls after HFD plus Dox feeding for 7 days. Equal loading control was demonstrated by α-β-actin antibody (n = 3 per group. Student’s t-test , *, p < 0.05, **, p < 0.01). (F) Oxygen consumption rate (OCR) in sWAT measured by seahorse machine. The sWAT was collected from VEGF Tg and their littermate control mice after 7 days DOX induction. The injection time and final concentrations of the different compounds used for the assay were shown at the above of the curves. Results were represented as mean° S.E.M., n=3 per group. Student’s t-test , *, p < 0.05). (G) Bioenergetic parameters inferred from OCR traces. Results were represented as mean° S.E.M., n=3 per group. Student’s t-test , *, p < 0.05, ***, p<0.001).

Article Snippet: For immunofluorescence (IF) staining, sections were stained with a primary antibody against COX IV (1:500, Cell Signaling Technologies) followed by a donkey α-rabbit Alexa Fluor 488-conjugated or donkey α-goat Alexa Fluor 647-conjugated secondary antibodies (1.5 μg/mL, Jackson Immuno-Research Laboratories, West Grove, PA).

Techniques: Staining, Western Blot, Control, Injection

Journal: bioRxiv

Article Title: Iron addicted colorectal cancers exploit Heme-Complex II axis to resist oxidative cell death

doi: 10.1101/2025.11.05.686813

Figure Lengend Snippet: A) Schematic showing the xenograft tumor model with subcutaneous implantation. B) Tumor weight from B) C) Lipid ROS are measured by C11-BODIPY581/591 in EPCAM positive cells from the tumors from B). D) Schematic showing breeding strategy to generate tamoxifen inducible intestine specific SDHC deficient mice. E) Western blot images showing cellular and mitochondrial protein expression of SDHC 5days post tamoxifen induction via 100mg/kg body weight I.P. injection in WT ( Sdhc +/+), Het ( Sdhc +/-) and KO ( Sdhc -/-) mice. F) Representative H&E images of Sdhc F/F and Sdhc ΔIE mice fed with 350 ppm or 1000ppm iron containing chow for 7 days. G) Inflammatory histology scored for small intestine and colon of Sdhc F/F and Sdhc ΔIE mice fed with 350 ppm or 1000ppm iron containing chow for 7 days. H) Representative immunohistochemistry images showing accumulation of 4 hydroxynonenal (4HNE) in small intestine and colon of Sdhc F/F and Sdhc ΔIE mice fed with 350 ppm or 1000ppm iron containing chow for 7 days. I) Schematic showing treatment protocol for colitis associated cancer model using AOM/DSS in Sdhc F/F (WT) and Sdhc Het IE (Het) mice. J) Tumor number measured in mice from I). K) Tumor burden measured in mice from I) L) Lipid ROS measured by C11-BODIPY581/591 in EPCAM positive epithelial cells derived from I). M) Schematic showing role of heme-SDHC-CoQ axis in mitigating iron induced cell death in CRC cells. All data are mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test for (B) and (G) and t test for (C), (J), (K) and (L). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Followed by blocking with 5% goat serum in PBS and sections were then probed with primary antibody against SDHC (Proteintech, 14575-AP) or 4HNE (R&D Systems, MAB3249-SP) diluted at 1:500 in the blocking solution.

Techniques: Western Blot, Expressing, Injection, Immunohistochemistry, Derivative Assay

Journal: bioRxiv

Article Title: Neuropathology in an α-synuclein preformed fibril mouse model occurs independent of the Parkinson’s disease-linked lysosomal ATP13A2 protein

doi: 10.1101/2024.08.07.607077

Figure Lengend Snippet: ( A , D , G , J , and M ) Representative images of pS129-α-synuclein immunostaining at 6 MPI in WT or ATP13A2 KO mice, from the contralateral and ipsilateral (injected) hemispheres. Scale bar: 200 µm. Digital pathology quantitation of pS129-α-synuclein immunostaining in the ( B ) forebrain, ( E ) cortex, ( H ) striatum, ( K ) amygdala, and ( N ) substantia nigra at 6 MPI. Data are expressed as the % area occupied by pS129-α-synuclein immunostaining in the contralateral or ipsilateral hemisphere, with bars representing the mean ± SEM, and individual data points for each animal ( n = 6–7 animals/group). * P <0.05 or ** P <0.01 by paired Student’s t -test comparing contralateral and ipsilateral hemispheres within the same genotype as indicated, and no significance between genotypes by two-way ANOVA with Tukey’s multiple comparisons test. ( C , F , I , L , and O ) Ipsilateral to contralateral ratio of % area of pS129-α-synuclein immunostaining for each animal within each brain region, with bars representing mean ± SEM for each genotype. P >0.05 by unpaired Student’s t -test.

Article Snippet: Primary antibodies against pS129-α-synuclein (ab51253; Abcam), tyrosine hydroxylase (TH; N300-109; Novus Biological), Iba1 (019-19741; Fujifilm Wako Chemical USA), and GFAP (ab227761; Abcam) were used.

Techniques: Immunostaining, Injection, Quantitation Assay

Journal: bioRxiv

Article Title: Unravelling biological processess and EGFR pathway regulation by the receptor-like protein tyrosine phosphatase PTPRH in non-small cell lung cancer

doi: 10.1101/2024.06.13.598886

Figure Lengend Snippet: H2023 and H23 PTPRH knockout cell lines transduced with BioID2-HA-only, PTPRH WT-BioID2-HA, or mock (control) were validated for expression of the constructs and their subcellular localization using immunoblot and immunofluorescence, respectively. A) Immunoblot against the HA-tag for mock (n=1/cell line), BioID2-only (n=2/cell line), and PTPRH WT-BioID2-HA (n=2/cell line) H2023 and H23 KO cells. Bands matching the correct molecular size for BioID2-HA and PTPRH WT-BioID2-HA are indicated by the arrows. B) Immunofluorescence was performed in all groups using antibodies against the HA-tag which are shown in magenta. DAPI staining is shown in blue. Representative cells showing localization of PTPRH WT-BioID2-HA in the plasma membrane and/or cytosol are indicated by arrows. Images are representative fields using 40x magnification, scale bar = 400 pixels. Abbreviations : ab = antibody; IB= immunoblot.

Article Snippet: The coverslips were then incubated with primary antibodies against BioID2 (1:5000, BioFront #BID2-CP-100) or HA tag (1:800, Cell Signaling #3724) diluted in 1% BSA/ PBS-Tween 0.1% for one hour in a humidified chamber at RT.

Techniques: Knock-Out, Transduction, Control, Expressing, Construct, Western Blot, Immunofluorescence, Staining, Membrane

Journal: bioRxiv

Article Title: Unravelling biological processess and EGFR pathway regulation by the receptor-like protein tyrosine phosphatase PTPRH in non-small cell lung cancer

doi: 10.1101/2024.06.13.598886

Figure Lengend Snippet: H2023 and H23 PTPRH knockout cell lines transduced with BioID2-HA-only, PTPRH WT-BioID2-HA, or mock (control) were stained with antibody against the BioID2 tag subcellular localization of protein is shown in green (immunofluorescence). DAPI staining is shown in blue. Representative cells showing localization of PTPRH WT-BioID2-HA in the plasma membrane and/or cytosol are indicated by arrows. Images are representative fields using 40x magnification, scale bar = 400 pixels. Abbreviations : ab = antibody.

Article Snippet: The coverslips were then incubated with primary antibodies against BioID2 (1:5000, BioFront #BID2-CP-100) or HA tag (1:800, Cell Signaling #3724) diluted in 1% BSA/ PBS-Tween 0.1% for one hour in a humidified chamber at RT.

Techniques: Knock-Out, Transduction, Control, Staining, Immunofluorescence, Membrane

Journal: bioRxiv

Article Title: Unravelling biological processess and EGFR pathway regulation by the receptor-like protein tyrosine phosphatase PTPRH in non-small cell lung cancer

doi: 10.1101/2024.06.13.598886

Figure Lengend Snippet: H2023 and H23 PTPRH KO cell lines transduced with BioID2-HA-only, PTPRH WT-BioID2-HA, or mock (negative control) were serum starved for 24h, followed by stimulation with FBS and biotin for 18h. A) Immunoblot against biotin showing the presence of biotinylated proteins in the BioID2-only and PTPRH WT-BioID2-HA groups, with some bands (arrows) showing up exclusively in the PTPRH-WT-BioID2-HA groups. B) Venn diagram of biotinylated proteins identified in each PTPRH WT-BioID2-HA transduced cell line (n=1/cell line) by mass spectrometry. These proteins present a minimum of 3-fold increase in normalized total precursor intensity compared to their respective BioID2-only transduced cell line. C) Candidate PTPRH interactors (n=48) were screened for the presence of reported phospho sites in the PhosphoSitePlus database. The percentage of proteins containing at least one pY or pS/pT sites are shown. D) STRING analysis of candidate PTPRH interactors (n=58). Abbreviations : IB = immunoblot; pY = phospho-Tyrosine; pS/pT = phospho-Serine/phospho-Threonine.

Article Snippet: The coverslips were then incubated with primary antibodies against BioID2 (1:5000, BioFront #BID2-CP-100) or HA tag (1:800, Cell Signaling #3724) diluted in 1% BSA/ PBS-Tween 0.1% for one hour in a humidified chamber at RT.

Techniques: Transduction, Negative Control, Western Blot, Mass Spectrometry

Journal: bioRxiv

Article Title: NaBC1 boron transporter enables myoblast response to substrate rigidity via fibronectin-binding integrins

doi: 10.1101/2024.06.14.599051

Figure Lengend Snippet: A: Quantification of myogenic differentiation in C2C12 myoblasts seeded on PAAm hydrogels of different stiffnesses, functionalized with fibronectin (FN) and stimulated with soluble boron (B) (0.59 and 1.47 mM). n = 10 images from 3 different biological replicates. B: Quantification of myogenic differentiation in C2C12 myoblasts seeded on PAAm hydrogels of different stiffnesses, functionalized with laminin-111 and stimulated with soluble B ions (0.59 and 1.47 mM). n = 10 images from 3 different biological replicates. C: Quantification of myogenic differentiation in NaBC1-KO C2C12 myoblasts, cultured as described in A. n = 10 images from 3 different biological replicates. D: Quantification of myogenic differentiation in C2C12 myoblasts transfected with the VD1 plasmid and cultured as described in A. n = 10 images from 3 different biological replicates. E: Representative images of myogenic differentiation in C2C12 myoblasts, cultured as described in A. Magenta: sarcomeric α-actinin; Cyan: DAPI. Scale bar: 100 µm. Myotubes were counted when three or more cell nuclei were aligned. Data are represented as Mean ± Standard Deviation, and differences are considered significant for p ≤ 0.05 using two-way ANOVAs (Tukey’s multiple comparisons tests) for multiple comparisons. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001

Article Snippet: For myogenic differentiation studies, mouse monoclonal primary antibody against sarcomeric α-actinin (1:200, Abcam) and rabbit anti-mouse Cy3-conjugated secondary antibody (1:200, Jackson Immunoresearch) were used.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Standard Deviation

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a , lmmunohistochemical analysis of prefrontal cortex sections from an individual with FTLD-TDP Type C (individual 1) using an antibody against pS409/410 TDP-43 (brown). Scale bar, 50 µm. b, lmmuno-EM analysis of filament extracts from the prefrontal cortex of an individual with FTLD-TDP Type C (individual 1) using an antibody against pS409/410 TDP-43 and a 10 nm gold-conjugated secondary antibody (black dots). Scale bars, 100 nm.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques:

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a , Cryo-EM reconstructions of filaments from FTLD-TDP Type C individual 1 with two alternative conformations of the TDP-43 glycine-rich region (indicated with arrows), shown as central slices perpendicular to the helical axis. Scale bars, 2 nm. b, Fourier shell correlation (FSC) curves for the two independently-refined cryo-EM half-maps (black lines); for the refined atomic model against the cryo-EM density map (magenta); for the atomic model shaken and refined using the first half-map against the first half-map (cyan); and for the same atomic model against the second half-map (yellow). FSC thresholds of 0.143 (black dashed line) and 0.5 (magenta dashed line) are shown. c, Local resolution estimates for the cryo-EM reconstructions. d, Cryo-EM reconstructions viewed along the helical axis. Scale bar, 1 nm. e,f, Views of the cryo-EM reconstructions and atomic models showing representative densities for ordered solvent (red arrows) (f) and main chain oxygen atoms in 13-strands, which reveal the chirality of the map.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Cryo-EM Sample Prep, Solvent

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a , Cryo-EM reconstruction of the left-handed filaments of ANXA11 and TDP-43 from FTLD-TDP Type C, shown parallel to the helical axis. b, Identification of TDP-43 and ANXA11 chains in the ordered filament fold. ANXA11 was identified by deriving a sequence motif directly from well-resolved amino acid side chain densities in the cryo-EM reconstruction (see Methods). c, Cryo-EM reconstruction and atomic model of the filaments, shown for single TDP-43 and ANXA11 chains perpendicular to the helical axis. The green arrow indicates an isolated peptide consistent with TDP-43 residues N352-G357. Buried ordered solvent is indicated with red dots. d and e, Domain organisation of TDP-43 (d) and ANXA11 (e). DIX, dishevelled and axin domain; N, nuclear localisation signal; RRM, RNA-recognition motif; LCD, low-complexity domain; ANX, annexin repeat. The black lines indicate the regions that form the filament fold. f and g, Amino acid sequence alignment of the secondary structure elements of the TDP-43 (f) and ANXA11 (g) chains. Arrows indicate -strands. The sequences that form the interface between TDP-43 and ANXA11 are underlined. a-c, Cryo-EM density for TDP-43 is in grey and ANXA11 is in yellow. b, c, f and g, The TDP-43 glycine-rich (G284-G310, magenta), hydrophobic (M311-S342, white) and QIN-rich (Q343-Q345, green) regions are highlighted. ANXA11 is shown in orange.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Cryo-EM Sample Prep, Sequencing, Isolation, Solvent

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a , Schematic of the secondary structure elements of the homotypic TDP-43 filament folds of ALS and FTLD-TDP Type A and B, and the heterotypic TDP-43 and ANXA11 fold of FTLD-TDP Type C. Side chains for R293 are shown. Alternative local conformations of the FTLD-TDP Type A and C folds are transparent. b and c, Amino acid sequence alignment of the secondary structure elements of TDP-43 (b) and ANXA11 (c) in the filament folds. Arrows indicate -strands. a-c, The TDP-43 glycine-rich (G282-G310, magenta), hydrophobic (M311-S342, white) and QIN-rich (Q343-Q360, green) regions are highlighted. ANXA11 is shown in orange. R293 is indicated with a blue dot.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: Double labelling immuno-EM analysis of filament extracts from the prefrontal cortex of an individual with FTLD-TDP Type C (individual 2) using antibodies against pS409/410 TDP-43 and N-terminal ANXA11 (residues 1-180) using 10 nm and 6 nm gold-conjugated secondary antibodies (black), respectively. The filaments label for both ANXA11 and TDP-43. Scale bars, 500 nm.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques:

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a , Secondary structure of the heteromeric filament fold of FTLD-TDP Type C, shown for single ANXA11 and TDP-43 molecules perpendicular to the helical axis. b, Atomic model of the filament fold depicting hydrogen bonding (dashed cyan lines), shown for three ANXA11 and TDP-43 molecules perpendicular to the helical axis. c, Hydrophobicity of the filament fold, from most hydrophilic (teal) to most hydrophobic (yellow), shown for single ANXA11 and TDP-43 molecules perpendicular to the helical axis. d, Atomic model of filament fold, shown for single ANXA11 and TDP-43 molecules aligned with the helical axis. a, b and d, The TDP-43 glycine-rich (G284-G310, magenta), hydrophobic (M311-S342, white) and QIN-rich (Q343-Q345, green) regions are highlighted. ANXA11 is shown in orange. a and c, The layers of the ANXA and TDP-43 chains are indicated with arrows.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques:

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a and b , Overlay of the cryo-EM reconstruction (a) and hydrophobicity surface plot (yellow, most hydrophobic; teal, least hydrophobic) (b) with the atomic model of the filaments, focussed on the interface between ANXA11 and TDP-43 and shown for single ANXA11 and TDP-43 chains perpendicular to the helical axis. Cryo-EM density for TDP-43 is in grey and ANXA11 is in yellow. Buried ordered solvent is indicated with a red dot. c, Atomic model of the interface between ANXA11 and TDP-43, shown for three molecular layers perpedicular to the helical axis.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Cryo-EM Sample Prep, Solvent

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a , Cryo-EM reconstruction and atomic model of heteromeric amyloid filaments from FTLD-TDP Type C with an alternative conformation of the glycine-rich region, shown for single ANXA11 and TDP-43 molecules perpendicular to the helical axis. Cryo-EM density for TDP-43 is in grey and ANXA11 is in yellow. Buried ordered solvent is indicated with red dots. b, Overlay of the atomic models of filaments with the alternative conformation of the glycine-rich region with the main conformation (transparent). c, Amino acid sequence alignment of the secondary structure elements of TDP-43 in the filaments. Arrows indicate (3-strands. C1, main conformation; C2, alternative conformation. d, Alignment of TDP-43 residues N295-R293 from the atomic models of FTLD-TDP Type C (pink) and Type A (cyan) filaments. a-c, The TDP-43 glycine-rich (G282-G310, magenta), hydrophobic (M311-S342, white) and QIN-rich (Q343-Q345, green) regions are highlighted. ANXA11 is show in orange.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Cryo-EM Sample Prep, Solvent, Sequencing

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a and b , lmmunoblot analysis of filament extracts from the prefrontal cortex of individuals with FTLD-TDP Types A (2), B (2) and C (6) using antibodies against N-terminal ANXA11 (residues 1-180) (a) and pS409/410 TDP-43 (b). A ∼22 kDa ANXA11 NTF (white arrow), as well as a minor population of full length ANXA11 (black arrow), are observed for all individuals with FTLD-TDP Type C, but not for individuals with FTLD-TDP Types A and B. Full length TDP-43 (black arrow) and TDP-43 CTFs (black line) are observed for all individuals. c, lmmunohistochemical analysis of prefrontal cortex sections from four individuals with FTLD-TDP Type C using antibodies against pS409/410 TDP-43 and N-terminal ANXA11 (residues 1-180). Individual images for TDP-43 and ANXA11 are shown in greyscale to facilitate comparison, in addition to a merged image showing TDP-43 (green), ANXA11 (magenta) and DAPI (blue) staining. ANXA11 and TDP-43 co-localise with inclusions. Additional immunohistochemical analysis is shown in and . Scale bar, 20 µm.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Comparison, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: a-c , lmmunohistochemical analysis of prefrontal cortex sections from individuals with FTLD-TDP Type C (a), Type A (b) and Type B (c) using antibodies against pS409/410 TDP-43 and N-terminal ANXA11 (residues 1-180). Individual images for TDP-43 and ANXA11 are shown in greyscale to facilitate comparison, in addition to a merged image showing TDP-43 (green), ANXA11 (magenta) and DAPI (blue) staining. ANXA11 and TDP-43 colocalise with inclusions in the individuals with FTLD-TDP Type C, but only TDP-43 co-localises with the inclusions in the individuals with FTLD-TDP Types A and B. Additional immunohistochemical analysis is shown in and Extended Data Fig. 9. Scale bar, 40 µm.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Comparison, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

doi: 10.1101/2024.06.25.600403

Figure Lengend Snippet: lmmunohistochemical analysis of fascia dentata sections from three individuals with FTLD-TDP Type C using antibodies against pS409/410 TDP-43 and N-terminal ANXA11 (residues 1-180). Individual images for TDP-43 and ANXA11 are shown in greyscale to facilitate comparison, in addition to a merged image showing TDP-43 (green), ANXA11 (magenta) and DAPI (blue) staining. ANXA11 and TDP-43 colocalise with inclusions. Additional immunohistochemical analysis is shown in and Extended Data Fig. 8. Scale bar, 40 µm.

Article Snippet: For colourimetric immunohistochemistry, sections were incubated overnight with a primary antibody against pS409/S410 TDP-43 (CosmoBio CAC-TIP-PTD-M01A, 1:1,000) in blocking buffer.

Techniques: Comparison, Staining, Immunohistochemical staining

Journal: bioRxiv

Article Title: KLF7-Regulated ITGA2 as a Therapeutic Target for Inhibiting Oral Cancer Stem Cells

doi: 10.1101/2024.11.04.621805

Figure Lengend Snippet: A The log2-transformed fold change (FC) in RNA levels between the stem-like cluster with the rest of the clusters and the stem-related pathway with the rest of the pathway. B Immunoblot assay of ITGA2, phosphor-ERK1/2, total-ERK1/2, phosphor-AKT, total-AKT, phosphor-YAP1, total-YAP1 protein levels in CAL27 and HSC3 cells after stable silencing ITGA2. C Representative immunofluorescent image showing the expression of YAP1 and DAPI in wt and stable silencing ITGA2 cells. D The sequence and domain in ITGA2 protein. E Co-IP analysis in CAL27 and HSC3 cells transduced with ITGA2 OE and ITGA2 mut plasmid. F Immunoblot assay of phosphor-ERK1/2, total-ERK1/2, phosphor-AKT, total-AKT, phosphor-YAP1, total-YAP1 protein levels in CAL27 and HSC3 cells after transduced with ITGA2 OE and ITGA2 mut plasmid. G Representative immunofluorescent image showing the expression of YAP1 and DAPI in CAL27 and HSC3 cells after transduced with ITGA2 OE and ITGA2 mut plasmid.

Article Snippet: After washing and blocking, the primary antibody against YAP1 (66900-1-Ig, Proteintech) was incubated overnight.

Techniques: Transformation Assay, Western Blot, Expressing, Sequencing, Co-Immunoprecipitation Assay, Transduction, Plasmid Preparation

Journal: bioRxiv

Article Title: KLF7-Regulated ITGA2 as a Therapeutic Target for Inhibiting Oral Cancer Stem Cells

doi: 10.1101/2024.11.04.621805

Figure Lengend Snippet: A The molecular formula of TC-I 15. B Co-IP analysis in CAL27 and HSC3 cells treated with DMSO and TC-I 15. C CAL27 cells were intracardially injected into mice. Seven days later, TC-I 15 was injected (20 mg/kg) into mice via vein every third day for 1 month. D Representative images showing the xenograft model in CA27 cells treated with DMSO or TC-I 15(n = 5 per group) and the growth of tumor grafts was shown. E Immunoblot assay of phosphor-ERK1/2, total-ERK1/2, phosphor-AKT, total-AKT, phosphor-YAP1, and total-YAP1 protein levels in xenograft tissue. F Representative FACS plots and quantification of CD133+cells in the xenograft tissues. G CAL27 cells were intracardially injected into mice. Seven days later, TC-I 15 (20 mg/kg) and plastin(5 mg/kg) were injected into nude mice via vein every third day for 1 month. H Representative images showing the xenograft model in CA27 cells treated with DMSO or TC-I 15(n = 5 per group) and the growth of tumor grafts were shown.

Article Snippet: After washing and blocking, the primary antibody against YAP1 (66900-1-Ig, Proteintech) was incubated overnight.

Techniques: Co-Immunoprecipitation Assay, Injection, Western Blot