Lamp1

Following isolation and plating, cells were subjected to immunohistochemical staining and microscopic imaging to assess the presence and activation state of TFEB (Bethyl Laboratories, Montgomery, TX) LAMP1 (ThermoFisher Scientific, Waltham, MA ), and LC3 (Sigma-Aldrich, St. Louis, MO) at dilutions of 1:5000, 1:500, and 1:200, respectively, following the manufacturers immunofluorescence histochemistry protocols. Immunohistochemical staining of the macrophage antigen F4/80 (Abcam, Cambridge, UK) was performed at a 1:500 dilution. The secondary antibody was Alexa-Fluor 488 (1:500 dilution) (Abcam, Cambridge, UK). Immunohistochemistry of TUNEL (R&D Systems, Minneapolis, MN) was performed using manufacturer’s protocol for tissue cryosections. After staining, cells or tissues were imaged using a Nikon Eclipse Ti inverted microscope (Nikon Instruments, Melville, NY) and Nikon DS-U3 camera (Nikon Instruments) and Photometrics CoolSnap MYO camera system (Photometrics, Tucscon, AZ), under control of Nikon NIS-Elements AR Software (Nikon Instruments). Illumination for fluorescence imaging is generated using the X-Cite 120Q Widefield Fluorescence Microscope Excitation Light Source (Excelitas Technology, Waltham, MA).

BMDCs were seeded in confocal dishes (Mattek) at a final concentration of 1×10⁵ cells/mL in RPMI-complete media. The following day, cells were treated with 10 μg/mL GRD for 5 minutes and 15 minutes at 37C. Cells were carefully washed with PBS and fixed with 2% PFA for 10 minutes at room temperature in the dark. Fixed cells were permeabilized with SAP buffer (2% saponin in PBS) for 1 hour at 37C. Following permeabilization, BMDCs were intracellularly stained with antibodies to mouse TLR7, MyD88, EEA1, or LAMP1 (Thermo Scientific), followed by staining with secondary antibodies fluorescently labeled with AlexaFluor488 or AlexFluor550 (Invitrogen). Cells were washed 3 times with PBS. Finally, DNA was stained with 1:1000 dilution of DAPI (Invitrogen) for 10 minutes at RT. Cells were visualized using a Nikon confocal microscope and analysis performed using ZEN software. Data analysis and processing of images was conducted using ImageJ software (NIH).

For cell culture experiments, cells were grown on sterile, gelatin-coated glass cover slips and were fixed at 24 h after the first passage with 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilized and background blocked with 0.3% Triton X-100 + 10% NGS in PBS for 45 min at room temperature. Primary antibodies were diluted in 1% NGS + 0.3% Triton X-100 + 1% BSA in PBS and incubated on slides overnight at 4°C. The primary antibodies and dilutions used were as follows: LAMP-1 (1:1000, Cat# 14–1071-82, Invitrogen, CA, USA), MCP-1 (1:500, Cat# MA5–17040, Invitrogen), IGF-1 (1:250, Cat# PAJ-27207, Invitrogen), and IGFBP-2 (1:250, Cat# MAB797, R&D Systems, NE, USA). Slides were incubated with appropriate secondary antibodies at a 1:300 dilution (AlexaFluors, Thermo Fisher Scientific). Samples stained for actin were incubated with ActinGreen 488 Ready Probe (Cat# R37110, Invitrogen). Slides were mounted with mounting medium containing DAPI nuclear counterstain (Cat# S36938, Thermo Fisher Scientific). Slides with secondary but no primary antibodies were used as negative background controls.