Chloroquine

Male homozygous hpa-TG mice, in which the human heparanase gene is driven by a constitutive β-actin promoter in a BALB/c genetic background, were applied [38 (link)]. Male wild-type (wt) BALB/c mice, 10–12 weeks old, were purchased from Harlan Laboratories (Jerusalem, Israel) and served as controls. Mice were maintained under conventional pathogen-free conditions, in a temperature-controlled room, and fed with standard mouse chow and tap water ad libitum. The study protocol was approved by the Technion Animal Inspection Committee (IL-0240217). All experiments were performed in accordance with the institutional guidelines and regulations. hpa-TG or wt mice were randomly assigned to the following experimental groups, 5 mice each: C—animals injected with vehicle (PBS) serving as control; A—experimental Adriamycin-induced nephropathy (10 mg/kg, i.v.), sacrificed after 3 days; Q—mice injected with chloroquine (40 mg/kg, i.p., Sigma), 7 h before being sacrificed; and AQ—mice injected with both Adriamycin and chloroquine.

Chloroquine was purchased from Sigma-Aldrich. Dihydroartemisinin and piperaquine were kindly provided by Mangalam Drugs and Organics Ltd., Mumbai, India. Stock solutions of Chloroquine and piperaquine were prepared in distilled water, dihydroartemisinin in pure methanol (Merck, Darmstadt, Germany). Drug concentration ranges were chosen based on the average IC50 values available in the literature^{10 (link),29 (link)–33 (link),35 ,51 –54 (link)}. Doubling concentrations ranging from 6 to 100 nM for Chloroquine, and 6 to 486 nM for piperaquine, 0.12 to 32 nM for dihydroartemisinin were tested.

HeLa cells (European Cell Culture Collection) were cultured in minimum essential medium with Earle’s salts (MEM EBS; BioConcept, 1‐31F01‐I), supplemented with 10% FCS (GE Healthcare), 100 U penicillin (BioConcept), 100 μg/ml streptomycin (BioConcept), and 2 mM L-glutamine (BioConcept). Cells were cultured at 37 °C and 5% CO₂ and split using Accutase (Innovative Cell Technologies) diluted 1:2 in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4). Chloroquine treatment was performed in MEM complete supplemented with 10 μM Chloroquine (Sigma-Aldrich C6628).
Primary murine hepatocytes were isolated and cultured as described elsewhere^{3 (link)}. For infection of cells, salivary glands of infected Anopheles stephensi mosquitoes from 16–26 days after blood feeds were isolated and homogenized to release sporozoites. Sporozoites were incubated with cells in medium containing 25 μg/ml Amphotericin B (Amresco E437) for 2 h. Subsequently, cells were rinsed and incubated in MEM EBS complete containing 2.5 μg/ml Amphotericin B.

To study the potential involvement of autophagy pathways in calcium deposition, collagen deposition, MMP-2, RANKL secretion by B. abortus infection in MC3T3-E1 cells, wortmannin or E64 plus leupeptin, bafilomycin A1, or chloroquine (Sigma-Aldrich de Argentina S.A.) were added 2 h before the beginning of infection. wortmannin was used at a concentration of 10 μM, E64 plus leupeptin were used at a concentration of 20 μM, bafilomycin A1 was used at a concentration of 200 nM and chloroquine (Sigma-Aldrich de Argentina S.A.) was used at a concentration of 50 μM (Mizushima et al., 2010 (link)).

For the analysis
of the progression of cells through cell cycle phases and sub-G1 peak
identification, 24 h after plating, cells were treated with the different
compounds. After 72 h, both floating and adherent cells were collected
and fixed with cold 70% ethanol. At least 24 h after fixation, cells
were incubated with 0.025 mg/mL Propidium Iodide for 15 min at room
temperature and analyzed. For Annexin V analysis, 24 h after plating,
cells were treated with 50 and 100 μM of the compounds. In the
case of chloroquine experiments, 10 μM chloroquine (Sigma-Aldrich,
A9165) was added 30 min before compounds. 72 h after treatment, both
floating and adherent cells were collected and stained with an Annexin
V-FITC apoptosis detection kit (ENZO #ALX-850-020-KI). Samples were
incubated 15 min in the dark at rt, and 10,000 events were then analyzed.

HEK293 cells grown in DMEM‐Glutamax (Gibco/Thermo Fisher Scientific) supplemented with 10% FBS (Sigma‐Aldrich) were transfected with calcium phosphate. Media was replaced with fresh media supplemented with 25 μM chloroquine (Sigma‐Aldrich) 1 h before transfection, and again with fresh media without chloroquine 4–6 h after transfection. Forty‐eight hours after transfection, cells were lysed with RIPA buffer supplemented with protease inhibitors. For co‐secretion assays (Figs 1, 3B and E, and EV1, EV2, EV3), media was replaced by serum‐free FreeStyle 293 media (Gibco/Thermo Fisher Scientific). The media was harvested 48 h after transfection and centrifuged for 10 min, 1,500 × g, and 4°C to remove cell debris. Protein A Sepharose (4B; Thermo Fisher Scientific) washed three times in PBS buffer was added to each sample (50 µl) and incubated for 2 h at 4°C. Beads were treated with heparinases as described below. For experiments with membrane‐bound proteins (Figs 2C and D and 3G), HEK293 cells were lysed with RIPA buffer supplemented with protease inhibitors 48 h after transfection. Cell lysates were diluted in IP buffer (100 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 20 mM Tris, pH 7.4) and incubated with covalently coupled anti‐HA (Sigma‐Aldrich A2095) or anti‐V5 (Sigma‐Aldrich A7345) beads overnight at 4°C. Beads were treated with heparinases as described below.