Journal: bioRxiv

Article Title: Programmable Lipid Nanoparticle Targeting via Corona Engineering

doi: 10.64898/2026.02.27.708523

Figure Lengend Snippet: ApoE mutants reduce ApoE-mediated LNP uptake and liver accumulation. (a) Schematic of dApoE precoating and haPCSK9 pretreatment strategies for modulating LNP biodistribution. (b) Representative fluorescence microscopy images showing uptake of GFP-loaded LNPs in HEK293FT cells after incubation with media containing wild-type (WT) ApoE or single, double, triple, and five-residue combinatorial ApoE mutants. Progressive combination of receptor-binding domain mutations resulted in increasingly strong inhibition of ApoE-mediated uptake, with the five-residue mutant exhibiting the most pronounced blockade. Scale bar, 100 µm. (c) Quantification of the LNP uptake by GFP plate-read measurements, confirming that while individual mutations partially reduced uptake, combinatorial mutations produced progressively stronger inhibition, with the five-residue ApoE mutant showing maximal suppression of LNP internalization. (d) Uptake of LNPs in HEK293FT and AML-12 (e) hepatocytes using purified proteins, comparing WT ApoE, three-residue ApoE mutant (dApoE-3m), five-residue ApoE mutant (dApoE-5m), and haPCSK9 pretreatment. Both dApoE and haPCSK9 robustly suppress ApoE-dependent uptake. (f) Proteomic analysis of LNP–protein corona composition showing that dApoE precoating stabilizes the corona and maintains mutant ApoE enrichment even after subsequent serum exposure, whereas LNPs incubated with 10% mouse serum alone recruit a broader range of serum proteins. (g) Serum competition assays demonstrating enhanced uptake with WT ApoE and persistent inhibition by dApoE at higher concentrations, consistent with LC-MS findings on corona stability. (h) In vivo bioluminescence images after 6h of LNP injection (0.3 mg/kg RNA) showing reduced hepatic accumulation with dApoE precoating (25× relative to LNP mRNA) or haPCSK9 pretreatment (40 µg per mouse, administered 15 min before LNP injection). (i) Quantification of bioluminescence across major organs (liver, spleen, kidneys, heart, lungs), showing selective reduction in liver uptake without redistribution to other tissues. All data in this figure are mean ± SEM (n=3).

Article Snippet: HEK293FT and AML-12 cells were purchased from ATCC.

Techniques: Fluorescence, Microscopy, Incubation, Residue, Binding Assay, Inhibition, Mutagenesis, Produced, Purification, Liquid Chromatography with Mass Spectroscopy, In Vivo, Injection

Journal: Microbiology Spectrum

Article Title: Novel nucleoside analogs exhibit potent intracellular and in vivo activities against Mycobacterium avium

doi: 10.1128/spectrum.02160-25

Figure Lengend Snippet: Effects of MCCB-04-35 and MCCB-04-37 on host cell viability and M. avium survival. ( A ) Cytotoxicity of MCCB-04-35 and MCCB-04-37 in AML12 cells. Data are presented as the percentage of viable cells relative to those of the untreated control. ( B ) Intracellular survival of M. avium in BMDMs following treatment with MCCB-04-35 and MCCB-04-37. The number of viable intracellular M. avium cells (CFU/mL) was quantified 24-h post-treatment. ( C ) Extracellular survival of M. avium in BMDMs following treatment with MCCB-04-35 and MCCB-04-37. The number of viable intracellular M. avium cells (CFU/mL) was quantified 24 h after treatment. Data are presented as mean ± SD from independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (one-way ANOVA).

Article Snippet: AML12 cells (CRL-2254; ATCC, Manassas, VA, USA), a murine hepatocyte cell line, were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen).

Techniques: Control

Journal: bioRxiv

Article Title: Rab32/Rab38-positive Lysosome-Related Organelle degrades lipid droplet in hepatocytes by microautophagy

doi: 10.64898/2026.02.13.705687

Figure Lengend Snippet: A, B . Representative immunofluorescence images of AML12 cells at low (30%) and high (80%) confluence stained for Rab32 or Rab38 (green) and LAMP1 (red). Nuclei were stained with DAPI (blue). Arrowheads indicate colocalization of Rab32/38 with LAMP1. Scale bars, 5 μm (low confluence) and 7.5 μm (high confluence). C. Representative live-cell images of GFP-Rab32 or GFP-Rab38 with Strawberry-Rab7 in AML12 cells. Scale bar, 5 μm. D. Representative live-cell images of GFP-Rab32 or GFP-Rab38 with Strawberry-Rab5 in AML12 cells. Scale bar, 5 μm. E. Quantification of colocalization between GFP-Rab38-positive LROs (>3 μm²) and Strawberry-Rab7 or Strawberry-Rab5 (n = 5 random fields per condition). Statistical significance was determined using a one-sample t-test ( p < 0.0001). Scale bar, 5 μm. F. Representative immunofluorescence images showing GFP-Rab32 with endogenous Rab38 and GFP-Rab38 with endogenous Rab32. Scale bar, 5 μm.

Article Snippet: Murine AML12 hepatocyte cells (ATCC, CRL-2254) were maintained in DMEM/F-12 supplemented with HEPES buffering system (11330032, Invitrogen; 500 mL), 10% fetal bovine serum (FBS; F7524, Sigma-Aldrich), 1× Penicillin-Streptomycin Solution (100×stock; 168-23191, Fujifilm Wako) and 1× Insulin-Transferrin-Selenium Solution (ITS-G) (100×stock; 41400045, Invitrogen) and 40 ng/ml Dexamethasone (≥98%, HPLC; D1756, Sigma-Aldrich) at 37 °C in a humidified 5% CO2 incubator.

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Rab32/Rab38-positive Lysosome-Related Organelle degrades lipid droplet in hepatocytes by microautophagy

doi: 10.64898/2026.02.13.705687

Figure Lengend Snippet: A. Representative DIC and dextran staining images of AML12 cells at low (30%) and high (80%) confluence. Scale bar, 25 μm. B. Quantification of vacuole (> 3 μm²) number per cell. Data are mean ± SD (low, n = 19; high, n = 15). Statistical significance was determined using the Mann-Whitney test (p = 0.0415). C. Immunoblotting of Rab32 and Rab38 in AML12 cells at ∼30%, ∼60%, and ∼80% confluence. Tubulin was used as a loading control. D. Quantification of Rab32 and Rab38 protein levels relative to tubulin. Bars represent mean ± SD from three independent experiments. p values were determined by one-sample t -test; Rab32: 30% vs 60% ( p = 0.0561), 30% vs 80% ( p = 0.0215). Rab38: 30% vs 60% ( p = 0.1924), 30% vs 80% ( p = 0.0389). E. Immunoblot analysis of Rab32 and Rab38 expression in control, shRab32, shRab38, and Rab32/38 double knockdown (DKD) AML12 cells. Tubulin was used as a loading control. A nonspecific band at ∼50 kDa was detected with the anti-Rab32 antibody. F. Representative DIC and dextran staining images of WT and DKD AML12 cells at high confluence. Scale bar, 10 μm. G. Quantification of vacuole number (>3 μm²) per field in WT and DKD cells (n = 10 random fields per condition, 5 cells per field). Data are mean ± SD. Statistical significance was determined using the Mann-Whitney test ( p = 0.0071). H. Representative live-cell imaging of dextran-stained vacuoles in AML12 cells expressing GFP control, GFP-Rab32, or GFP-Rab38 at low confluence. Scale bar, 25 μm. I. Quantification of vacuole number (>3 μm²) per single cell (n = 25 cells per condition). Data are mean ± SD. Statistical significance was determined using the Mann-Whitney test (GFP vs GFP-Rab32, p = 0.0304; GFP vs GFP-Rab38, p < 0.0001).

Article Snippet: Murine AML12 hepatocyte cells (ATCC, CRL-2254) were maintained in DMEM/F-12 supplemented with HEPES buffering system (11330032, Invitrogen; 500 mL), 10% fetal bovine serum (FBS; F7524, Sigma-Aldrich), 1× Penicillin-Streptomycin Solution (100×stock; 168-23191, Fujifilm Wako) and 1× Insulin-Transferrin-Selenium Solution (ITS-G) (100×stock; 41400045, Invitrogen) and 40 ng/ml Dexamethasone (≥98%, HPLC; D1756, Sigma-Aldrich) at 37 °C in a humidified 5% CO2 incubator.

Techniques: Staining, MANN-WHITNEY, Western Blot, Control, Expressing, Knockdown, Live Cell Imaging, Single Cell

Journal: bioRxiv

Article Title: Rab32/Rab38-positive Lysosome-Related Organelle degrades lipid droplet in hepatocytes by microautophagy

doi: 10.64898/2026.02.13.705687

Figure Lengend Snippet: A. Representative live-cell fluorescence images of AML12 cells expressing GFP-Rab32 and LAMP1-RFP, stained with Lipi-Blue (white), under vehicle, 50 µM orlistat, or 50 µM orlistat and 20 nM Bafilomycin A1 (BafA1) for 24 h. Arrowheads indicate lipid droplets enclosed by Rab32/38-positive LROs. Scale bar, 2.5 µm. B. Representative images of AML12 cells treated with 50 µM orlistat and co-expressing mStrawberry-Atg4BC74A and GFP-Rab38. Lipi-blue marks lipid droplets. Dashed lines outline cell boundaries. Arrowheads indicate LDs engulfed by Rab38-positive LROs, indicating macroautophagy-independent LD engulfment. Scale bar, 5 µm.

Article Snippet: Murine AML12 hepatocyte cells (ATCC, CRL-2254) were maintained in DMEM/F-12 supplemented with HEPES buffering system (11330032, Invitrogen; 500 mL), 10% fetal bovine serum (FBS; F7524, Sigma-Aldrich), 1× Penicillin-Streptomycin Solution (100×stock; 168-23191, Fujifilm Wako) and 1× Insulin-Transferrin-Selenium Solution (ITS-G) (100×stock; 41400045, Invitrogen) and 40 ng/ml Dexamethasone (≥98%, HPLC; D1756, Sigma-Aldrich) at 37 °C in a humidified 5% CO2 incubator.

Techniques: Fluorescence, Expressing, Staining

Journal: bioRxiv

Article Title: Rab32/Rab38-positive Lysosome-Related Organelle degrades lipid droplet in hepatocytes by microautophagy

doi: 10.64898/2026.02.13.705687

Figure Lengend Snippet: A. Representative fluorescence images of AML12 control (-), sh Rab32 , sh Rab38 , and Rab32/38 double-knockdown (DKD) cells stained with Hoechst (blue) and Lipi Deep-Red (red). Scale bars, 10 µm. B. Quantification of total LD area per cell in control (-), sh Rab32 , sh Rab38 , and DKD cells. Data are presented as mean ± SD (control, n = 15; sh Rab32 , n = 15; sh Rab38 , n = 13; DKD, n = 15). Statistical significance was determined using Brown-Forsythe ANOVA with multiple comparisons (control vs sh Rab32 , p < 0.0001; control vs sh Rab38 , p < 0.0001; control vs DKD, p = 0.0003). C. Representative images of orlistat-treated control (-), sh Rab32 , sh Rab38 , and DKD cells stained with Lipi-Blue (green) and Lysotracker (red). Arrowheads indicate LDs surrounded by lysosomes, whereas asterisks indicate LDs showing impaired lysosomal engagement in DKD cells. Insets show magnified views. Scale bar, 5 µm. D. Quantification of the fraction of LDs overlapping with lysosomal structures in each genotype. Data are mean ± SD from randomly acquired fields (control, n = 6; sh Rab32 , n = 5; sh Rab38 , n = 5; DKD, n = 6). Statistical significance was determined using ordinary one-way ANOVA with multiple comparisons (control vs sh Rab32 , ns, p = 0.993; control vs shRab38, p = 0.0181; control vs DKD, p = 0.0064). E. Representative images of WT and DKD AML12 cells expressing LAMP1-RFP (red) and stained with Lipi-blue (cyan) following orlistat treatment. Insets show zoomed regions highlighting LD-lysosome associations. Scale bar, 10 µm. F. Quantification of the proportion of LDs localized inside LAMP1-RFP positive structures relative to total LDs in WT and DKD cells. Data are mean ± SD from 10 cells in each group. Statistical significance was determined using the Mann-Whitney test ( p < 0.0001).

Article Snippet: Murine AML12 hepatocyte cells (ATCC, CRL-2254) were maintained in DMEM/F-12 supplemented with HEPES buffering system (11330032, Invitrogen; 500 mL), 10% fetal bovine serum (FBS; F7524, Sigma-Aldrich), 1× Penicillin-Streptomycin Solution (100×stock; 168-23191, Fujifilm Wako) and 1× Insulin-Transferrin-Selenium Solution (ITS-G) (100×stock; 41400045, Invitrogen) and 40 ng/ml Dexamethasone (≥98%, HPLC; D1756, Sigma-Aldrich) at 37 °C in a humidified 5% CO2 incubator.

Techniques: Fluorescence, Control, Knockdown, Staining, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Rab32/Rab38-positive Lysosome-Related Organelle degrades lipid droplet in hepatocytes by microautophagy

doi: 10.64898/2026.02.13.705687

Figure Lengend Snippet: A. Representative live-cell fluorescence images of AML12 cells expressing GFP-Rab38 together with the PI3P probe mCherry-2xFYVE or the PI(3,5)P₂ probe PX-SnxA GV -GCC-mCherry, stained with Lipi-Blue. Cells were treated with vehicle, 50 µM orlistat, or 50 µM orlistat and 1 µM SAR405 for 24 h. Scale bar, 2 µm. B. Representative live-cell fluorescence images of AML12 scramble, sh Vps4a , and sh Vps4b cells transduced with a GFP-Rab38-expressing retrovirus and stained with Lipi-DeepRed. Cells were treated with vehicle (DMSO) or orlistat (200 µM, 24 h) prior to imaging. Scale bar, 10 µm. C. Quantification of Lipi-DeepRed average intensity in scramble, sh Vps4a , and sh Vps4b AML12 cells transduced with GFP-Rab38 and treated with vehicle (DMSO) or orlistat. Data are presented as mean ± SD (n ≥ 20 single cells per condition). Statistical significance was determined by two-way ANOVA with multiple-comparisons testing. **** p < 0.0001; ns, not significant.

Article Snippet: Murine AML12 hepatocyte cells (ATCC, CRL-2254) were maintained in DMEM/F-12 supplemented with HEPES buffering system (11330032, Invitrogen; 500 mL), 10% fetal bovine serum (FBS; F7524, Sigma-Aldrich), 1× Penicillin-Streptomycin Solution (100×stock; 168-23191, Fujifilm Wako) and 1× Insulin-Transferrin-Selenium Solution (ITS-G) (100×stock; 41400045, Invitrogen) and 40 ng/ml Dexamethasone (≥98%, HPLC; D1756, Sigma-Aldrich) at 37 °C in a humidified 5% CO2 incubator.

Techniques: Fluorescence, Expressing, Staining, Transduction, Imaging