Journal: bioRxiv

Article Title: Breast cancer interactions with osteoclasts generate osteoclast-tumor hybrid-like cells through dynamic non-canonical cell fusion and cell-in-cell processes

doi: 10.64898/2026.04.05.716538

Figure Lengend Snippet: a , Schematic diagram showing co-culture fusion assay between osteoclast precursors and solid tumor cell lines. b , Representative live cell images of an osteoclast-tumor hybrid (yellow outline) formed between RAW264.7-GFP osteoclasts (green) and 4T1-H2B-mRFP breast cancer cells (red). Tumor nuclei expressed H2B-mRFP (white arrow) within osteoclast cytoplasm. Scale bar, 100 μm. c , Schematic representation for Cre reporter lentiviral plasmid construct containing a floxed DsRed-E2 (stop cassette), followed by a downstream GFP. Cre mediated recombination excises the stop cassette and allows GFP to be expressed. d,e , Representative immunofluorescence images of BMM-derived osteoclasts co-cultured with EO771 Cre reporter. ( d ) Primary osteoclast-tumor hybrids (BMO Hybrid 1 & 2) (yellow outline) were formed in the co-culture assay with tumor nuclei identified based on RFP (DsRed-E2) fluorescence and round nuclei morphology (red arrows). ( e ) Co-cultured primary cells osteoclasts and tumor cells expressed Cre-recombinant product, GFP. Scale bars, 100 μm. f , Representative image showing a developing RAW264.7-GFP osteoclast-4T1-H2B-mRFP tumor hybrid surrounded by pre-osteoclasts and tumor cells. Tumor cells and pre-osteoclasts that were adjacent to the hybrid (blue arrows) had round morphologies while tumor cells located further away from the hybrid were epithelial-shaped (yellow arrow). g-i , Fluorescent time-lapse video showing cell fusion events between RAW264.7-GFP and 4T1-H2B-mRFP at various stages of osteoclast differentiation. ( g ) Tumor cell (yellow arrow) converged with other pre-osteoclasts to form a multinucleated osteoclast. Video stills were acquired from Movie S1 ( h ) Two osteoclast-tumor hybrids (yellow arrow) fused with each other. Video stills were acquired from Movie S2. ( i ) Individual tumor cells (yellow arrow) and pre-osteoclasts (white arrow) fused into an existing hybrid cell. Video stills were acquired from Movie S3.

Article Snippet: RAW264.7, 4T1, EO771, B16F10, and CT26 cell lines were obtained from ATCC.

Techniques: Co-Culture Assay, Single Vesicle Fusion Assay, Plasmid Preparation, Construct, Immunofluorescence, Derivative Assay, Cell Culture, Co-culture Assay, Fluorescence, Recombinant

Journal: bioRxiv

Article Title: Breast cancer interactions with osteoclasts generate osteoclast-tumor hybrid-like cells through dynamic non-canonical cell fusion and cell-in-cell processes

doi: 10.64898/2026.04.05.716538

Figure Lengend Snippet: a , Schematic diagram showing co-culture fusion assay between osteoclast precursors and solid tumor cell lines. b , Representative live cell images of a hybrid-like cell (yellow outline) formed between RAW264.7-GFP osteoclasts (green) and 4T1-H2B-mRFP breast cancer cells (red). Tumor nuclei expressed H2B-mRFP (white arrow) within osteoclast cytoplasm. Scale bar, 100 μm. c , Representative live cell images of a hybrid-like cell (yellow outline) formed between a primary bone-marrow-derived osteoclast (actin outline) and 4T1-H2B-mRFP breast cancer cells (magenta). Scale bar, 100 μm. d-f , Fluorescent time-lapse video showing cell fusion events between RAW264.7-GFP and 4T1-H2B-mRFP at various stages of osteoclast differentiation. ( d ) Tumor cell (yellow arrow) converged with other pre-osteoclasts to form a multinucleated osteoclast. Video stills were acquired from Movie S1 ( e ) Two osteoclast-tumor hybrids (yellow arrow) fused with each other. Video stills were acquired from Movie S2. ( f ) Individual tumor cells (yellow arrow) and pre-osteoclasts (white arrow) fused into an existing hybrid cell. Video stills were acquired from Movie S3. g, Live cell image showing RAW-246.7 GFP and 4T1-H2B-mRFP in distinct morphologies. Tumor and pre-osteoclast cells adjacent to an osteoclast displayed round cell shape (blue arrows) while tumor cells in growing colony were epithelial-shaped (yellow arrow). h, Representative immunofluorescent images for RAW264.7-GFP osteoclast-tumor hybrid-like cells stained for H3K4me2 transcriptional activity. Hybrids 1 and 2 contained tumor nuclei that fully colocalized with H3K4me2.

Article Snippet: RAW264.7, 4T1, EO771, B16F10, and CT26 cell lines were obtained from ATCC.

Techniques: Co-Culture Assay, Single Vesicle Fusion Assay, Derivative Assay, Staining, Activity Assay

Journal: bioRxiv

Article Title: Breast cancer interactions with osteoclasts generate osteoclast-tumor hybrid-like cells through dynamic non-canonical cell fusion and cell-in-cell processes

doi: 10.64898/2026.04.05.716538

Figure Lengend Snippet: a,b, Fluorescent snapshot image for osteoclast-tumor hybrid-like cells formed between RAW264.7-GFP and other solid tumor cell lines. Hybrids containing tumor nuclei derived from (a) B16F10-H2B-mRFP melanoma and hybrids with tumor nuclei derived from (b) CT26-H2B-mRFP colon cancer.

Article Snippet: RAW264.7, 4T1, EO771, B16F10, and CT26 cell lines were obtained from ATCC.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Breast cancer interactions with osteoclasts generate osteoclast-tumor hybrid-like cells through dynamic non-canonical cell fusion and cell-in-cell processes

doi: 10.64898/2026.04.05.716538

Figure Lengend Snippet: a , Schematic diagram displaying live cell laser microdissection protocol on RAW264.7-GFP osteoclasts co-cultured with 4T1-H2B-mRFP breast cancer cells to excise hybrids and other cells of interest for downstream single cell sequencing. b , Representative fluorescent images for three osteoclast sample types with hybrids (OHC) (n = 10), non-hybrid osteoclasts from co-culture (OC) (n = 10), and non-hybrid osteoclasts from osteoclast-only culture (O) (n = 7) with osteoclasts highlighted in yellow outlines. c , Bar graph showing three DEGs comparison groups with OHC vs. O, OC vs. O, and OHC vs. OC. Upregulated, downregulated, and overall DEGs were displayed for each group. d , Heatmap displaying both upregulated and downregulated DEGs amongst the three osteoclast sample types. e , Venn diagram showing overlapping and unique overall DEGs amongst the three osteoclast comparison groups. f , PCA analysis plot for individual samples from three osteoclast sample types. g,h Gene ontology (GO) analyses for hybrids and non-hybrid osteoclasts on three comparison groups (OHC vs. O, OC vs. O, and OHC vs. OC). Upregulated gene functions on biological processes (BP), cellular components (CC), and molecular functions (MF) for ( g ) OHC vs. O, OC vs. O, and ( h ) OHC vs. OC. ( i ) Attenuated gene functions on BP, CC, and MF for OHC vs. O. Gene functions and their respective gene counts were shown. p.adjusted value < 0.05. j-l, KEGG pathway analyses for the same comparison groups. Top five pathways augmented in various categories for ( j ) OHC vs. O, ( k ) OC vs. O, and ( l ) OHC vs. OC. Pathways were shown in order of significance with following annotations: no asterisk – p.adjusted value & p value < 0.05, one asterisk (*) – p.adjusted value > 0.05 & p value < 0.05, two asterisks (**) – p. adjusted value & p value > 0.05. ( m ) Network analysis for significant pathways enriched in OHC vs. O (red), OC vs. O (blue), and both (yellow). p.adjusted value < 0.05.

Article Snippet: RAW264.7, 4T1, EO771, B16F10, and CT26 cell lines were obtained from ATCC.

Techniques: Laser Capture Microdissection, Cell Culture, Single Cell, Sequencing, Co-Culture Assay, Comparison

Journal: bioRxiv

Article Title: Breast cancer interactions with osteoclasts generate osteoclast-tumor hybrid-like cells through dynamic non-canonical cell fusion and cell-in-cell processes

doi: 10.64898/2026.04.05.716538

Figure Lengend Snippet: a, Schematic diagram showing two co-culture setups for RAW264.7 preosteoclasts and 4T1 breast cancer cells under indirect (paracrine) and direct contacts. b, Cytokine array membrane blots for the two conditions. Top ten cytokine factors (fold change) enhanced in either direct contact or paracrine interaction conditions compared to each other. Cytokines associated with pro-inflammation (red), anti-inflammation (green), and angiogenesis (blue) were labelled. No significance was shown for cytokine array data. c, qPCR analysis on 4T1 isolated from direct contact and paracrine conditions with 4T1 from regular culture as baseline control. d, Dorothea analysis on TUMOR_CO vs TUMOR_UT showing differentially activated transcription factors. e, Western blot analysis for RAW264.7 osteoclasts and 4T1 cells under various culture conditions: osteoclasts only - O, breast cancer cells only - BC, osteoclasts from direct co-culture - O (DC), breast cancer cells from direct co-culture - BC (DC), osteoclasts from paracrine co-culture – O (P), breast cancer cells from paracrine co-culture – BC (P). f, Schematic diagram depicting bone metastasis induction on Ctsk-Cre and wild type (WT) mice models using EO771 breast cancer and B16-F10 melanoma expressing DsRed-E2-to-GFP Cre reporter system. g, Fluorescent images for DsRed-E2 fluorescent detection on mice with successful bone metastasis induction using EO771 and B16F10 expressing Cre reporter. h,i, Flow cytometry analysis on gated single cells showing DsRed-E2+ and GFP+ cells in EO771 and B16-F10 bone metastases derived from Ctsk-Cre mice. Statistics for GFP+ Cre recombined cells (per 500000 events) for EO771 bone metastases in Ctsk-Cre (n = 8) & WT (n = 5), and EO771 bone marrow in Ctsk-Cre (n = 5) & WT (n = 4). Statistics for GFP+ Cre recombined cells (per 500000 events) for B16F10 bone metastases in Ctsk-Cre (n = 6) & WT (n = 4), and B16F10 bone marrow in Ctsk-Cre (n = 5) & WT (n = 3). j , GFP+ recombined cells in bone metastases versus extraskeletal metastases for EO771 model. Statistics for GFP+ Cre recombined cells (per 500000 events) in EO771 with bone metastases (n = 8) & extraskeletal metastases (n = 6). Data are shown as means ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: RAW264.7, 4T1, EO771, B16F10, and CT26 cell lines were obtained from ATCC.

Techniques: Co-Culture Assay, Membrane, Isolation, Control, Western Blot, Expressing, Flow Cytometry, Derivative Assay

Journal: bioRxiv

Article Title: Breast cancer interactions with osteoclasts generate osteoclast-tumor hybrid-like cells through dynamic non-canonical cell fusion and cell-in-cell processes

doi: 10.64898/2026.04.05.716538

Figure Lengend Snippet: a, Dorothea analyses for OHC vs. O and OC vs. O highlighted Yap1-dependent TEAD1 transcription factor expression in hybrid-like cells. b, RAW264.7 osteoclasts from direct co-culture with 4T1 breast cancer cells promoted Yap1 downstream gene activation (Amotl2, Ankrd1, Ctgf, Cyr6I, Itga2, Serpine1) and enhanced ECM-modifying factors (Fn1, Pcolce2, Col4a2, Col5a3). c, Tumor nuclei present in primary and RAW264.7 hybrid-like cells displayed nuclear colocalized Yap1. d, Hybrid-like cell containing tumor nucleus with colocalized Yap1 were located adjacent to osteoclast nuclei expressing low Yap1 signals. e, Hybrid-like cells stained for p21 cell cycle inhibitor with p21-colocalized nuclei indicated as p21-positive (+) and non-p21-colocalized nuclei indicated as p21-negative (-). f, OHC-level quantification of p21-negative tumor nuclei, plotted as the fraction of p21-negative tumor nuclei per individual OHC with Wilson 95% confidence intervals; significance versus 50% was assessed by one-sample Wilcoxon signed-rank test (p = 0.00632). g, Nucleus-level quantification of p21-negative tumor nuclei pooled across OHCs, plotted as the overall p21-negative fraction with Wilson 95% confidence intervals; significance versus 50% was assessed by exact binomial test (p = 0.617).

Article Snippet: RAW264.7, 4T1, EO771, B16F10, and CT26 cell lines were obtained from ATCC.

Techniques: Expressing, Co-Culture Assay, Activation Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Injectable Particulated Human Acellular Dermal Matrix Booster for Skin Restoration: An Integrated Randomized, Split-Face, Double-Blinded Clinical Trial and Preclinical Study

doi: 10.3390/ijms27052193

Figure Lengend Snippet: Anti-inflammatory properties of phADM in mouse macrophages. After 24 h of treatment with LPS (1 µg/mL), the production of IL-1β ( A ), TNF-α ( B ), IL-6 ( C ), and PGE-2 ( D ) in mouse macrophages significantly increased. Treatment with phADM (0.3–1%) successfully counteracted this increase. L-NMMA (100 µM) served as positive control. Data are presented as mean ± SD. ∗ comparison with negative control, ∗ p < 0.05. # comparison with LPS control, # p < 0.05. LPS, lipopolysaccharide. phADM, particulated human acellular dermal matrix. L-NMMA, N-monomethyl-L-arginine. IL, interleukin. TNF-α, tumor necrosis factor–alpha. PGE-2, prostaglandin E2.

Article Snippet: HDFs (Thermo Fisher Scientific, Waltham, MA, USA), used for the evaluation of anti-wrinkle efficacy; mouse melanoma cells (B16F10, American Type Culture Collection, Manassas, VA, USA), used for the evaluation of skin-whitening efficacy; and mouse macrophages (RAW 264.7, American Type Culture Collection), used for the evaluation of anti-inflammatory efficacy, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco).

Techniques: Positive Control, Comparison, Negative Control, Control

Journal: Food Chemistry: X

Article Title: Elderberry ( Sambucus nigra L.) extracts promote anti-inflammatory and cellular antioxidant activity

doi: 10.1016/j.fochx.2022.100437

Figure Lengend Snippet: Anti-inflammatory activity promoted by elderberry extracts in LPS-stimulated RAW 264.7 cells, assessed by NO release quantification. Effect of elderberry crude extract, E ( A ); rich-phenolic extract, M ( B ); and non-phenolic extract, H ( C ); from Sabugueiro (So), Sabugueira (Sa) and Bastardeira (Ba) cultivars. Results are presented as mean ± SD (n = 3 independent experiments, each one in quadruplicates), set as % of control (LPS-exposed cells). Significant statistical differences between the control and samples are denoted by an “*”, and between samples at same concentration by an “*” over square brackets, when ( p < 0.05).

Article Snippet: Caco-2 (human colon adenocarcinoma, CLS, Eppelheim, Germany), HepG2 (human hepatoma cell line; ATCC, Rockville, MD, USA) and RAW 264.7 (mouse macrophages, Abelson murine leukemia virus-induced tumor, CLS, Germany) cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium) containing 25 mM glucose supplemented with 10 % (v/v) fetal bovine serum (FBS; Gibco, Life technologies), 2 mM l -glutamine (Gibco, Life technologies), 100 U/mL penicillin (Life technologies) and 100 μg/mL streptomycin (Life technologies) at 37 °C in a normal atmosphere of 5 % CO 2 in air and were handled as described ( ).

Techniques: Activity Assay, Control, Concentration Assay

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Schematic of Lrrk2 domain organization indicating the position of the ROC P-loop substitution T1348N. The catalytic core is shown in black; scaffold domains are indicated in grey. ( B-B’ ) Immunoblots of endogenous Lrrk2 steady-state levels in murine RAW 264.7 macrophage cell lines (WT, Lrrk2 T1348N, and Lrrk2 KO), with β-actin as loading control ( B ), and corresponding quantification of Lrrk2 abundance normalized to WT within each independent experiment ( n = 3, B’ ). ( C-C’ ) Schematic of the protocol for differentiation of primary bone marrow-derived macrophages (BMDMs) from WT and Lrrk2 T1348N KI mice using GM-CSF (granulocyte-macrophage colony-stimulating factor; 50 ng/mL) for 7 days (representative brightfield image for illustration purposes only, C ), and immunoblots of endogenous Lrrk2 steady-state levels in BMDMs, with β-actin as loading control ( C’ ). ( D - E ) Immunoblots of Lrrk2-mediated phosphorylation of Rab10 at Thr73 (pRab10 T73) in RAW 264.7 cells ( D ) treated with vehicle (CTRL), chloroquine (CQ, 50 μM, 2 h) or the Lrrk2 inhibitor MLi-2 (0.1 μM, 90 min), and BMDMs ( E ) under vehicle (CTRL) or CQ (50 μM, 2 h). β-actin is used as loading control. ( F-I ) Representative immunofluorescence images of RAW 264.7 WT, Lrrk2 T1348N and Lrrk2 KO cells treated with vehicle or L-leucyl-L-leucine methyl ester (LLOMe; 1 mM, 30 min), showing stimulus-dependent puncta formation/recruitment of Lrrk2 (left), Rab8A (middle) and Rab10 (right) ( F ). Scale bars 10 μm. Quantification shows the number of Lrrk2-positive ( G ), Rab8A-positive ( H ) and Rab10-positive ( I ) puncta per cell under vehicle and LLOMe conditions. For each biological replicate ( n = 3), multiple fields were quantified and averaged to yield one value per replicate. Data are presented as mean ± SEM unless otherwise stated; solid dots represent independent biological replicates (mean of technical replicates, shown as open dots, within each experiment). Statistical significance: ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical test: ( B’ ) two-tailed one-sample t test. ( G-I ) two-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Western Blot, Control, Derivative Assay, Phospho-proteomics, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) RT-qPCR analysis of Lrrk2 transcript levels in murine RAW 264.7 cells (WT, Lrrk2 T1348N, and Lrrk2 KO), normalized and expressed relative to WT ( n = 5 replicates). ( B-B’ ) Cycloheximide (CHX) chase in RAW 264.7 WT and Lrrk2 T1348N cells (CHX, 1 ng/μL; time points as indicated), with immunoblot detection of Lrrk2 and β-actin as loading control ( B ). Densitometric quantification shows Lrrk2/β-actin expressed as a fraction of the t = 0 signal within each genotype ( B’ ). ( C-C’ ) Immunoblots of Lrrk2 levels in RAW 264.7 WT and Lrrk2 T1348N cells following inhibition of major degradation pathways (MG132, 20 μM, 16 h for proteasome inhibition; CQ, 50 μM, 16 h for lysosomal neutralization), with β-actin as loading control ( C ). p62 and LC3B are shown as pathway response markers. Quantification of Lrrk2 abundance across treatments is expressed as fold-change relative to vehicle (CTRL) within each genotype ( n = 3 independent experiments, C’ ). ( D-D’ ) Solubility fractionation of WT and T1348N Lrrk2, assessing distribution between Triton X-100 -soluble and -insoluble fractions ( D ); quantification shows soluble Lrrk2 over total, normalized to WT within each independent experiment ( n = 3, D’ ). Data are presented as mean ± SEM; solid dots represent independent biological replicates (mean of technical replicates, shown as open dots, within each experiment). Statistical significance: ns, not significant; ** p < 0.01, **** p < 0.0001. Statistical tests: ( A, D’ ) one-sample t test; ( B’ ) two-way ANOVA with Tukey’s multiple comparisons; ( C’ ) two-way ANOVA with Šídák’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Quantitative RT-PCR, Western Blot, Control, Inhibition, Neutralization, Solubility, Fractionation

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Cellular thermal shift assay (CETSA)/thermal denaturation profiling of endogenous Lrrk2 in RAW 264.7 WT and T1348N cells across the indicated temperature range, assessed by immunoblotting. ( A’ ) Quantification of Lrrk2 band intensity from ( A ), expressed as percentage of the 4°C condition and plotted as a function of temperature. Data are presented as individual measurements and are representative of two independent experiments; curves are shown as fitted trends.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Thermal Shift Assay, Western Blot

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A-A’ ) Immunoblots of p62 levels in RAW 264.7 macrophage cell lines (WT, Lrrk2 T1348N, and Lrrk2 KO), with β-actin as loading control ( A ), and corresponding quantification relative to WT ( n = 5 independent experiments, A’ ). ( B-B’ ) Immunoblots of p62 levels in primary BMDMs from WT and Lrrk2 T1348N KI mice ( B ), with quantification relative to WT ( n = 3 independent experiments, B’ ). ( C-C’ ) Autophagic flux assessment in RAW 264.7 WT, Lrrk2 T1348N, and Lrrk2 KO cells expressing the tandem mCherry-GFP-LC3 reporter under vehicle or CQ treatment (50 µM, 16 h). Representative images show GFP, mCherry, and merged channels ( C ); quantification shows the fraction of GFP-mCherry-positive puncta normalized to total mCherry puncta per cell ( n = 3 independent experiments, C’ ). (D) Immunoblots of Lrrk2 and p62 levels in brain, lung, and kidney lysates from WT and Lrrk2 T1348N KI mice collected at 1, 6, and 12 months (pooled samples per condition), with β-actin as loading control. ( E-E’’ ) Immunoblots of individual kidney lysates from WT and T1348N mice at 1 month ( E ), 6 months ( E’ ), and 12 months, with β-actin as loading control ( E’’ ). ( F-G ) Densitometric quantification of Lrrk2 ( F ) and p62 ( G ) levels in kidneys from ( E-E’’ ), expressed relative to WT at each age ( n = 5 WT mice, n = 6 Lrrk2 T1348N mice). Data are presented as mean ± SEM; solid dots represent independent biological replicates. Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical tests: ( A’ ) one-way ANOVA with Tukey’s multiple comparisons; ( B’ ) unpaired two-tailed Student’s t test; ( C’ ) Kruskal-Wallis with Dunn’s multiple comparisons; ( F, G ) two-way ANOVA with Šídák’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Western Blot, Control, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Schematic of Lrrk2 domain organization indicating the position of the ROC P-loop substitution T1348N. The catalytic core is shown in black; scaffold domains are indicated in grey. ( B-B’ ) Immunoblots of endogenous Lrrk2 steady-state levels in murine RAW 264.7 macrophage cell lines (WT, Lrrk2 T1348N, and Lrrk2 KO), with β-actin as loading control ( B ), and corresponding quantification of Lrrk2 abundance normalized to WT within each independent experiment ( n = 3, B’ ). ( C-C’ ) Schematic of the protocol for differentiation of primary bone marrow-derived macrophages (BMDMs) from WT and Lrrk2 T1348N KI mice using GM-CSF (granulocyte-macrophage colony-stimulating factor; 50 ng/mL) for 7 days (representative brightfield image for illustration purposes only, C ), and immunoblots of endogenous Lrrk2 steady-state levels in BMDMs, with β-actin as loading control ( C’ ). ( D - E ) Immunoblots of Lrrk2-mediated phosphorylation of Rab10 at Thr73 (pRab10 T73) in RAW 264.7 cells ( D ) treated with vehicle (CTRL), chloroquine (CQ, 50 μM, 2 h) or the Lrrk2 inhibitor MLi-2 (0.1 μM, 90 min), and BMDMs ( E ) under vehicle (CTRL) or CQ (50 μM, 2 h). β-actin is used as loading control. ( F-I ) Representative immunofluorescence images of RAW 264.7 WT, Lrrk2 T1348N and Lrrk2 KO cells treated with vehicle or L-leucyl-L-leucine methyl ester (LLOMe; 1 mM, 30 min), showing stimulus-dependent puncta formation/recruitment of Lrrk2 (left), Rab8A (middle) and Rab10 (right) ( F ). Scale bars 10 μm. Quantification shows the number of Lrrk2-positive ( G ), Rab8A-positive ( H ) and Rab10-positive ( I ) puncta per cell under vehicle and LLOMe conditions. For each biological replicate ( n = 3), multiple fields were quantified and averaged to yield one value per replicate. Data are presented as mean ± SEM unless otherwise stated; solid dots represent independent biological replicates (mean of technical replicates, shown as open dots, within each experiment). Statistical significance: ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical test: ( B’ ) two-tailed one-sample t test. ( G-I ) two-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Western Blot, Control, Derivative Assay, Phospho-proteomics, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) RT-qPCR analysis of Lrrk2 transcript levels in murine RAW 264.7 cells (WT, Lrrk2 T1348N, and Lrrk2 KO), normalized and expressed relative to WT ( n = 5 replicates). ( B-B’ ) Cycloheximide (CHX) chase in RAW 264.7 WT and Lrrk2 T1348N cells (CHX, 1 ng/μL; time points as indicated), with immunoblot detection of Lrrk2 and β-actin as loading control ( B ). Densitometric quantification shows Lrrk2/β-actin expressed as a fraction of the t = 0 signal within each genotype ( B’ ). ( C-C’ ) Immunoblots of Lrrk2 levels in RAW 264.7 WT and Lrrk2 T1348N cells following inhibition of major degradation pathways (MG132, 20 μM, 16 h for proteasome inhibition; CQ, 50 μM, 16 h for lysosomal neutralization), with β-actin as loading control ( C ). p62 and LC3B are shown as pathway response markers. Quantification of Lrrk2 abundance across treatments is expressed as fold-change relative to vehicle (CTRL) within each genotype ( n = 3 independent experiments, C’ ). ( D-D’ ) Solubility fractionation of WT and T1348N Lrrk2, assessing distribution between Triton X-100 -soluble and -insoluble fractions ( D ); quantification shows soluble Lrrk2 over total, normalized to WT within each independent experiment ( n = 3, D’ ). Data are presented as mean ± SEM; solid dots represent independent biological replicates (mean of technical replicates, shown as open dots, within each experiment). Statistical significance: ns, not significant; ** p < 0.01, **** p < 0.0001. Statistical tests: ( A, D’ ) one-sample t test; ( B’ ) two-way ANOVA with Tukey’s multiple comparisons; ( C’ ) two-way ANOVA with Šídák’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Quantitative RT-PCR, Western Blot, Control, Inhibition, Neutralization, Solubility, Fractionation

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Cellular thermal shift assay (CETSA)/thermal denaturation profiling of endogenous Lrrk2 in RAW 264.7 WT and T1348N cells across the indicated temperature range, assessed by immunoblotting. ( A’ ) Quantification of Lrrk2 band intensity from ( A ), expressed as percentage of the 4°C condition and plotted as a function of temperature. Data are presented as individual measurements and are representative of two independent experiments; curves are shown as fitted trends.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Thermal Shift Assay, Western Blot

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Schematic overview of the AP-MS workflow: anti-Flag affinity purification of 3xFlag-LRRK2 WT or T1348N expressed in HEK-293T cells, followed by LC-MS/MS analysis. ( B-B’’’ ) Representative differential interactors of WT and T1348N LRRK2. Proteins are grouped into functional categories, expressed as fold-change relative to WT: chaperones ( B ), 14-3-3 proteins ( B’ ), cytoskeletal proteins ( B’’ ), ribosomal proteins ( B’’’ ). Each dot represents one quantified interactor. (C) Ranked plot of differential interaction strength for proteins quantified by AP-MS, shown as log 2 fold-change (T1348N/WT). Selected hits are highlighted, including TRIM32, molecular chaperones, and members of the 14-3-3 family. ( D-D’ ) Immunoblot validation of selected AP-MS hits in anti-Flag immunoprecipitates from HEK-293T cells expressing 3xFlag-LRRK2 WT or T1348N under control (CTRL), starvation (STARV, 16 h serum deprivation followed by 2 h in HBSS), or CQ treatment (50 μM, 16 h), including assessment of LRRK2 phosphorylation at Ser935, chaperones (Hsp90/Hsp70), TRIM32, and 14-3-3 proteins (γ and β) ( D ). Quantification of TRIM32 and Hsp70 co-purification, expressed as fold-change relative to WT within each condition ( n = 2 technical replicates, D’ ). Data are presented as mean ± SEM where quantified; symbols represent individual proteins ( B-C ) or technical replicates ( D’ ).

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Protein-Protein interactions, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Western Blot, Biomarker Discovery, Expressing, Control, Phospho-proteomics, Copurification

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A-A’ ) Immunoblots of p62 levels in RAW 264.7 macrophage cell lines (WT, Lrrk2 T1348N, and Lrrk2 KO), with β-actin as loading control ( A ), and corresponding quantification relative to WT ( n = 5 independent experiments, A’ ). ( B-B’ ) Immunoblots of p62 levels in primary BMDMs from WT and Lrrk2 T1348N KI mice ( B ), with quantification relative to WT ( n = 3 independent experiments, B’ ). ( C-C’ ) Autophagic flux assessment in RAW 264.7 WT, Lrrk2 T1348N, and Lrrk2 KO cells expressing the tandem mCherry-GFP-LC3 reporter under vehicle or CQ treatment (50 µM, 16 h). Representative images show GFP, mCherry, and merged channels ( C ); quantification shows the fraction of GFP-mCherry-positive puncta normalized to total mCherry puncta per cell ( n = 3 independent experiments, C’ ). (D) Immunoblots of Lrrk2 and p62 levels in brain, lung, and kidney lysates from WT and Lrrk2 T1348N KI mice collected at 1, 6, and 12 months (pooled samples per condition), with β-actin as loading control. ( E-E’’ ) Immunoblots of individual kidney lysates from WT and T1348N mice at 1 month ( E ), 6 months ( E’ ), and 12 months, with β-actin as loading control ( E’’ ). ( F-G ) Densitometric quantification of Lrrk2 ( F ) and p62 ( G ) levels in kidneys from ( E-E’’ ), expressed relative to WT at each age ( n = 5 WT mice, n = 6 Lrrk2 T1348N mice). Data are presented as mean ± SEM; solid dots represent independent biological replicates. Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical tests: ( A’ ) one-way ANOVA with Tukey’s multiple comparisons; ( B’ ) unpaired two-tailed Student’s t test; ( C’ ) Kruskal-Wallis with Dunn’s multiple comparisons; ( F, G ) two-way ANOVA with Šídák’s multiple comparisons.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Western Blot, Control, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of catalytic activity and impaired proteostasis in guanosine nucleotide-depleted LRRK2

doi: 10.64898/2026.04.21.719846

Figure Lengend Snippet: ( A ) Representative immunoblots of Lrrk2, Trim32, and p62 levels in kidney lysates from 12-month-old WT and T1348N mice, with β-actin as loading control. ( B-C’ ) Quantification of Trim32 ( B ) and p62 ( C ) normalized to β-actin and expressed relative to WT ( n = 5 WT mice, n = 6 Lrrk2 T1348N mice). p62 levels are also shown stratified by sex within the T1348N cohort (female n = 3 vs male n = 3, C’ ). (D) Representative immunofluorescence images of kidney sections from female WT and T1348N mice stained for Lamp1, p62, and Trim32, with nuclei counterstain. Boxed regions are shown as magnified insets. Scale bars 50 µm (main images) or 10 µm (crops), as indicated. (E) Quantification of p62-Lamp1 overlap (Pearson’s correlation coefficient) in kidney sections. ( F-H ) Quantification of p62 ( F ), Lamp1 ( G ), and Trim32 ( H ) signals by integrated density in kidney sections. Data are presented as mean ± SEM; dots represent individual mice. For immunofluorescence quantifications ( E - H ), 4 fields per mouse were quantified and averaged to generate one value per mouse ( n = 3 WT mice, n = 3 T1348N mice; see STAR Methods). Statistical significance: * p < 0.05; ** p < 0.01. Statistical tests: ( B-C’, E-H ) unpaired two-tailed Student’s t test.

Article Snippet: RAW 264.7 WT (SC-6003), Lrrk2 KO (SC-6004), and Lrrk2 T1348N KI (SC-6005) as well as HEK-293T cells were purchased from ATCC.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Two Tailed Test