To examine whether Otlip or any other O. turicata americanus salivary factors impact mammalian cytokine and chemokine expression, murine RAW macrophages were treated with recombinant GST (rGST) or GST-Otlip (rGST-Otlip) proteins (Supplementary Figure 1) or with lysates prepared from salivary glands isolated from unfed or fed nymphal ticks. RayBiotech cytokine protein array C3 kit was used in these assays (Supplementary Figure 2). Membranes that were spotted with antibodies against 62 different cytokines and chemokines were treated with supernatants collected from different treatment groups (Figure 1). One membrane was used per treatment group (Figures 1A–D). We detected differential signal intensities for the cytokines and chemokines after ~30 to 60 seconds exposure in all treatment groups (Figure 1). Some signal intensities were noted to be higher, and some were noted to be lower when different groups were compared (comparison was made between rGST or rGST-Otlip treatment groups or between unfed or fed salivary gland lysate treatment groups). The signal intensity data was collected from with Image-J software. The average of the duplicate signal intensities was calculated; the background was subtracted and normalized to the positive controls.

We then used Power BI software to quantify the levels of secreted cytokine and chemokine from murine macrophages treated with rGST/rGST-Otlip or unfed/fed salivary gland lysates. The cytokines with a Fold change value of 1 were considered having no differential expression. The bar graphs are arranged in a gradient showing the cytokine with the increased fold change at the top and gradually ending in the cytokine with the decreased fold-change at the bottom. The top five cytokines shown in the bar graph that were secreted from RAW macrophages at increased levels upon rGST-Otlip treatment when compared to the levels noted upon rGST treatment are FAS-ligand (5.6 fold), IL-1 beta (2.9 fold), LIX (2.7 fold), MIP-3 alpha (2.5 fold) and IGFBP3 (2.3 fold) (Figure 2A). The bottom five cytokines in the bar graph that were secreted at the decreased levels upon rGST-Otlip treatment when compared to levels noted upon GST treatment are GCSF (7.6 fold), TPO (7.1 fold), TIMP-1 (7.1 fold), RANTES (5.8 fold) and IL-3 (5.8 fold) (Figure 2A). Similar analysis was performed with data generated from treatment with unfed or fed tick salivary gland lysates. We noted increased levels of FAS-ligand (6.6 fold), IL-17A (5.5 fold), IGFBP3 (4.5 fold), MCP-1 (1.7 fold) and CXCL-16 (1.3 fold) upon treatment with fed tick salivary gland lysates compared to the levels noted upon treatment with unfed salivary gland lysates (Figure 2B). In addition, we noted decreased levels of L-selectin (4.5 fold), TECK (4.5 fold), MIP-3 alpha (4.3 fold), VCAM-1 (4.1 fold) and CD40 (3.5 fold) upon treatment with fed tick salivary gland lysates compared to the levels noted upon treatment with unfed tick salivary gland lysates (Figure 2B). We noted disturbed cell morphology (rounding up of cells) upon treatment with rGST-Otlip or fed tick salivary gland lysate compared to the morphology noted upon treatment with rGST or unfed tick salivary gland lysate, respectively (Supplementary Figure 3). Collectively, these results show that FAS-ligand and IGFBP3 were among the top 5 that are secreted from murine macrophages at increased levels upon treatment with rOtlip or with fed tick salivary gland lysates.

We further validated the protein array data by analyzing transcript levels upon treatment of murine macrophages with rGST/GST-Otlip or unfed/fed tick salivary gland lysates. RNA was extracted from these treated murine macrophages and processed for quantitative-real time PCR analysis (qRT-PCR analysis). Based on the cytokine protein array expression profile, we selected top three upregulated and bottom two downregulated genes in the rGST/rGST-Otlip or unfed/fed array tick salivary gland lysate treatment groups (Figure 3). The qRT-PCR analysis revealed significant (P<0.05) upregulation of fas-ligand (Figure 3A), lix (Figure 3B) and igfbp3 (Figure 3C) transcripts in macrophages treated with rGST-Otlip compared to the levels noted upon treatment with rGST control. In addition, we noted significant (P<0.05) downregulation of il-3 (Figure 3D) and gcsf (Figure 3E) transcripts upon treatment with rGST-Otlip compared to the levels noted upon treatment with rGST. Furthermore, treatment of murine macrophages with fed tick salivary gland lysates showed significant (P<0.05) upregulation of fas-ligand (Figure 3F), il-17 (Figure 3G) and igfbp3 (Figure 3H) transcripts compared to the levels noted upon treatment with unfed tick salivary gland lysates. No differences in the vcam-1 (Figure 3I) transcript levels in murine macrophages were noted between unfed or fed tick salivary gland lysates treated groups. We also noted that treatment of murine macrophages with fed tick salivary gland lysates showed significant (P<0.05) downregulation in teck transcripts compared to the levels noted upon treatment with unfed tick salivary gland lysates (Figure 3J). These qRT-PCR results validated some of the proteins noted to be differentially secreted from macrophages upon treatment with rGST-Otlip or fed tick salivary gland lysates.

The observation of changed cell morphology (Supplementary Figure 3), secretion of increased levels of IGFBP-3 (Figure 2) and increased igfbp3 transcripts in murine macrophages (Figure 3C) upon treatment with rGST-Otlip prompted us to further focus this study on this host molecule. IGFBP-3 binds to its receptor IGFBP-3R and is involved in inducing cell death (30–34). We therefore reasoned to test the effect of rGST-Otlip on igfbp-3r and apoptotic genes, and protein expression. qRT-PCR analysis showed significantly (P<0.05) higher transcript levels of igfbp-3r (Figure 4A) and the pro-apoptotic genes bax (Figure 4B), caspase-9 (Figure 4C) and caspase-3 (Figure 4D) upon treatment with rGST-Otlip compared to the transcript levels noted upon treatment with rGST control. In addition, we noted significantly (P<0.05) lower level of anti-apoptotic gene, bcl-2, upon treatment with rGST-Otlip compared to the levels noted upon treatment with rGST control (Figure 4E). Furthermore, immunoblotting analysis showed increased level of IGFBP-3 and Caspase-3 in macrophages upon treatment with rGST-Otlip compared to the levels noted upon rGST control treatment (Figure 4F; Supplementary Figure 4, 5). These results show that rGST-Otlip induces pro-apoptotic genes and downregulates anti-apoptotic gene in murine macrophages.

The expression levels of apoptotic genes were further quantified in murine macrophages upon treatment with salivary gland lysates generated from unfed or fed O. turicata americanus ticks. qRT-PCR analysis indicated significantly (P<0.05) higher expression levels of igfbp-3r (Figure 5A), and pro-apoptotic markers bax (Figure 5B), caspase-9 (Figure 5C) and caspase-3 (Figure 5D) transcripts in murine macrophages upon treatment with salivary gland lysates generated from fed ticks compared to the levels noted upon treatment with salivary gland lysates generated from unfed ticks. In addition, we noted that transcript level of anti-apoptotic gene bcl-2 (Figure 5E) were significantly (P<0.05) lower in murine macrophages upon treatment with salivary gland lysates generated from fed ticks compared to the levels noted upon treatment with salivary gland lysates generated from unfed ticks. Furthermore, immunoblotting analysis showed increased level of IGFBP-3 and Caspase-3 in macrophages upon treatment with salivary gland lysates generated from fed ticks compared to the levels noted upon treatment with salivary gland lysates generated from unfed ticks (Figure 5F; Supplementary Figures 4, 5). Collectively, these results support the observations noted with rGST-Otlip in inducing pro-apoptotic genes and reducing anti-apoptotic gene expression in murine macrophages.

The observation of induced pro-apoptotic and reduced anti-apoptotic gene expression and protein levels in murine macrophages prompted us to further investigate whether treatment with rGST-Otlip protein has any effect on cell viability. We performed Live/Dead assay to determine the cell death upon treatment of murine macrophages with rGST-Otlip. Brightfield and fluorescent microscopy imaging showed a higher number of live murine macrophage cells upon treatment with control rGST (Figure 6A) compared to the number noted upon treatment with rGST-Otlip (Figure 6A). In contrast, we noted higher number of dead murine macrophage cells upon treatment with rGST-Otlip (Figure 6A) compared to the number noted upon treatment with rGST control (Figure 6A). Furthermore, the MTT assay corroborated the Live/Dead imaging data where the number of viable murine macrophage cells were significantly (P<0.05) reduced upon treatment with rGST-Otlip compared to number of viable cells noted upon treatment with rGST control (Figure 6B). These results show that rGST-Otlip induces cell death in murine macrophages.

Since skin keratinocytes are a major part of the barrier between the tick-host interface and are among the first responders to tick bite (35), we wanted to investigate whether Otlip has any effects on apoptotic gene expression. We noted increased vacuolated cells upon treatment of human keratinocytic cell line, HaCaT cells, with rGST-Otlip compared to cells treated with rGST control (Supplementary Figure 6). qRT-PCR analysis showed significantly (P<0.05) higher expression of igfbp3 (Figure 7A) and caspase-3 (Figure 7B) transcripts in HaCaT cells upon treatment with rGST-Otlip compared to the levels noted upon treatment with rGST control. In addition, significantly (P<0.05) lower level of anti-apoptotic bcl-2 gene expression was noted upon treatment of HaCaT cells with rGST-Otlip compared to levels noted upon treatment with rGST control (Figure 7C). Furthermore, immunoblotting analysis supported the qRT-PCR results (Figure 7D; Supplementary Figures 7, 8). These results show that rGST-Otlip not only exert its effect on IGFBP3 and apoptotic signaling in macrophages but also in human keratinocytes.

We then performed Live/Dead assay to determine whether rGST-Otlip-mediated induction of apoptotic gene expression in HaCaT cells has any effect on cell viability. Microscopic analysis revealed increased number of viable HaCaT cells upon treatment with control rGST (Figure 8A) compared to the number of viable cells noted upon treatment with rGST-Otlip (Figure 8A). Like in murine macrophages, we noted increased number of dead HaCaT cells upon treatment with rGST-Otlip (Figure 8A) compared to the number of dead cells noted upon treatment with control rGST (Figure 8A). MTT assay further supported the Live/Dead imaging data where the number of viable HaCaT cells were significantly (P<0.05) reduced upon treatment with rGST-Otlip compared number of viable cells noted upon treatment with control rGST control (Figure 8B). Taken together, these results show that Otlip not only induces cell death in murine macrophages but also reveals similar effects in human keratinocytes.

For detection and visualization of the phenomenon of apoptosis elicited by tick Otlip protein in murine raw macrophages, we performed the TUNEL assay. Murine macrophages were treated with rGST control or rGST-Otlip and processed for TUNEL assay. Untreated macrophage cells were used as control. Based on the microscopic analysis, we noted higher numbers of TUNEL-positive macrophage cells upon treatment with control rGST-Otlip compared to the number noted upon treatment with rGST control or number noted in untreated cells (Figure 9A). Furthermore, we quantified the number of TUNEL-positive cells from several images and plotted data based on the percentage of TUNEL-positive cells from each group. The quantitative data shows significantly increased TUNEL-positive cells in murine macrophages upon treatment with rGST-Otlip compared to the number noted upon treatment with rGST control or number noted in untreated cells (Figure 9B). These data indicate that Otlip induces apoptosis in macrophages.

We then performed siRNA-mediated silencing experiments to further analyze whether Otlip directly mediates IGFBP-3-associated signaling to induce apoptosis. Murine macrophages were first treated with igfbp3-siRNA or scrambled siRNA followed by treatment with rGST-Otlip. qRT-PCR analysis revealed significantly (P<0.05) reduced expression of igfbp3 in igfbp3-siRNA-rGST-Otlip-treated murine macrophages compared to the levels noted in scrambled-siRNA-rGST-Otlip-treated cells (Figure 10A). In addition, we noted significantly (P<0.05) lower levels of caspase-3 transcripts (Figure 10B) and higher levels of bcl-2 transcripts in igfbp3-siRNA-rGST-Otlip-treated murine macrophages compared to the levels noted in scrambled-siRNA-rGST-Otlip-treated cells (Figure 10C). Furthermore, microscopic analysis showed increased cell death in scrambled-siRNA-rGST-Otlip-treated macrophages compared to igfbp3-siRNA-rGST-Otlip-treated cells (Supplementary Figure 9). Immunoblotting analysis supported qRT-PCR analysis (Figure 10D; Supplementary Figure 10). We noted lower levels of IGFBP3 and Caspase-3 proteins in igfbp3-siRNA-rGST-Otlip-treated murine macrophages compared to the levels noted in scrambled-siRNA-rGST-Otlip-treated cells (Figure 10D; Supplementary Figure 10). Taken together, these results indicate that O. turicata americanus tick lipocalin (Otlip) induces apoptosis in mammalian macrophages and keratinocytes by influencing IGFBP3-mediated signaling.

Laboratory-reared uninfected Ornithodoros turicata americanus ticks were used in this study. These ticks were provided by Dr. J.H. Oliver, Jr. from Georgia Southern University, Statesboro, GA, to Dr. Daniel Sonenshine at Old Dominion University (ODU), VA. The colony was maintained at ODU. A detailed description of these specimens is provided in our previous publications (52, 53). To generate fed nymphs, unfed nymphs were fed on naïve 6–8 weeks-old CD1 mice (Charles River Laboratories, USA). Fed repleted ticks were collected and processed for dissections of salivary glands. In addition, salivary gland tissues were also isolated from unfed nymphs. These tissues were dissected in 1X PBS and total protein lysates were prepared in complete RIPA lysis buffer (ThermoFisher Scienctific, USA). Protein concentration was measured using BCA protein assay kit (ThermoFisher Scientific, USA).

Tick feeding studies on animals was performed based on the approved Institutional animal care and use committee (IACUC) protocol (10–018) at Old Dominion University. During tick feeding, Acepromazine was used as a tranquilizer to minimize discomfort/anxiety in animals.

We previously identified and cloned the lipocalin-like molecule, otlip, from the soft ticks Ornithodoros turicata americanus (22). Escherichia coli clones were inoculated in 3 mL Luria Broth (LB) containing 50 microgram/mL of ampicillin and incubated overnight in a shaker. The overnight culture was used to inoculate a fresh 50 mL LB with ampicillin, and the cells were allowed to grow till optical density (OD)₆₀₀ between 0.5-0.6. The protein expression was induced by addition of 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and E. coli cells were allowed to grow at 37°C in a shaker for up to an hour or two for the OD₆₀₀ to reach 1-1.2. After this, the cells were centrifuged at 4200 rpm for 15 minutes. To these pelleted bacterial cells, 1 mL Lysis buffer containing 1X PBS, 0.2 mg/mL lysozyme, 10 mM beta mercaptoethanol, 1% Triton X-100 and with 1mM PMSF was added and incubated in a shaker at 37°C for one hour. In the next step, sonication was performed (for ~20 secs) with intervals of ice incubation and pulsing. After 5–6 repeated round of sonication, the culture was centrifuged at 15000 rpm for 15 mins at 4°C. The supernatant was collected and to this 0.5–1 mL glutathione resin (GBiosciences, USA) was added and kept overnight at 4°C in a rotor/shaker. Next day, the mixture was pelleted by centrifugation at 2000g for 5 min. The pellet was mixed with 1 mL of wash buffer and transferred to a 5 mL disposable polypropylene column (ThermoScientific, USA) and placed in a 15 mL tube. Following 3 washes (by centrifugation of the assembly at 2000g for 3 minutes), 1 ml elution buffer was added and incubated at room temperature for 5 minutes and centrifuged at 2000g for 3 minutes. This was repeated 3 times to get 3 ml of rGST or rGST-Otlip. In the next step, dialysis was performed overnight with 1X PBS using Slide-A-Lyzer dialysis cassettes (ThermoScientific, USA). The dialyzed solutions (containing rGST or rGST-Otlip) were filtered through 10-KDa cut-off filter for obtaining concentrated and purified recombinant rGST (~26 kDa) and rGST-Otlip (~32 kDa). Protein concentration of dialyzed recombinant proteins was measured using Pierce BCA protein assay kit (ThermoFisher Scientific, USA). The proteins were loaded onto 12% SDS-PAGE gel to check quality of the purified proteins (Supplementary Figure 1).

RAW 264.7 macrophage (ATCC, USA) and HaCaT cells (Fisher Scientific, USA) were seeded at a density of 1e5 cells per well in six replicates for each treatment in 12-well plates. Next day, these cells were treated with either 5 μg/mL/well of salivary gland lysates from unfed or fed ticks, or with rGST or rGST-Otlip. After 24 hours post treatment, cells were visualized under the microscope. In addition, cells were collected for RNA and protein extractions in RNA lysis buffer (BioRad, USA) or RIPA lysis buffer with protease inhibitor (ThermoFisher Scientific, USA), respectively. Cell culture supernatants were also collected and processed for Cytokine Array analysis.

Cytokine array analysis was performed using RayBiotech C-series Mouse Cytokine Antibody Array C3 (RayBiotech, USA) and with supernatants collected from RAW macrophages treated with salivary gland lysates from unfed or fed ticks or with rGST rGST-Otlip proteins. The Mouse Cytokine Array C3 (Ray Biotech Inc.) consisted of 62 different cytokine and chemokine antibodies spotted in duplicate onto a membrane. Four individual membranes were used in this study with one membrane/treatment group. All incubations and washes were performed under gentle rocking conditions at room temperature following manufacturer instructions. Care was taken to avoid appearance of bubbles on or between the membranes to ensure even distribution of the samples. Firstly, the membranes were blocked with 2 mL of blocking buffer and incubated at room temperature. Following aspiration of the buffer, 1 mL of supernatant (pooled from 6 replicates) was added to each array membrane and incubated overnight at 4°C. Next day, the samples were aspirated and 2 mL of 1X Wash Buffer I was added to each well and incubated for 5 minutes at room-temperature. This was repeated 2 more times for a total of 3 washes using fresh buffer and aspirating out the buffer completely each time. Similar wash steps were performed with 2 mL of 1X Wash Buffer II. In the next step, overnight incubation with biotinylated antibody cocktail was done under gentle rocking condition. Following this step, on the next day, similar wash steps with Wash Buffer I and II were repeated and then 2 mL of 1X HRP-Streptavidin was added to each well and incubated overnight at 4°C under gentle rocking condition. Next day, the solution was aspirated followed by washes with Wash Buffer I and II. The two individual membranes were placed side by side in a plastic protective folder and 500 µl of Detection Buffer (250 µl Detection Buffer C + 250 µl Detection Buffer D) was gently pipetted onto each membrane and incubated at room temperature for 2 minutes. Another plastic sheet was placed on top of the membranes by gently rolling the flexible plastic sheets such that the two membranes become sandwiched between the two plastic folders. The sandwiched array membranes were then imaged using chemiluminescence imaging system (BioRad, USA). Exposure times ranged from 30–60 seconds. The relative intensity densities indicate the change in the expression levels of the cytokines/chemokines. The intensities of the spots were quantified using Image-J software. Following subtraction of the negative control and normalization to the positive controls, integrated density value (IDV) for each cytokine in rGST-Otlip-treated or fed tick salivary gland lysate-treated samples was compared to the corresponding spot in the controls (rGST- or unfed-tick salivary gland lysate-treated samples). Fold-change values for all molecules in the cytokine array were calculated using IDVs and imported into Power BI (Microsoft, Redmond, WA, USA). Molecules were sorted in descending order based on their respective fold-change values, and a histography was generated with fold-change values used for the length and label for each bar. A color gradient was applied to the bars, with the neutral value (fold change = 1) shown in yellow, the most upregulated molecule in red, and the most downregulated molecule in blue.

Total RNA from RAW macrophage and HaCaT cell samples were generated using the Aurum Total RNA mini kit (Bio-Rad, USA) following the manufacturer’s instructions. RNA (200–250 ng) was converted to cDNA in a 10 µl reaction using BioRAD cDNA synthesis kit (BioRAD, USA) or QSCRIPT cDNA kit (QuantaBio/VWR, USA). One microliter of the generated cDNA was used as a template in each qRT-PCR reaction for quantifying transcript levels of cytokines/chemokines. Oligonucleotides used for quantifying mouse beta actin, IGFBP-3, IGFBP-3R, LIX, VCAM-1, TECK, IL-17, FAS ligand, G-CSF and IL-3 are published previously (54–63). Oligonucleotides used for quantifying human beta actin, IGFBP3, BCL-2 and Caspase-3 are previously published (11, 64–67). All other oligonucleotides used in this study are mentioned in Supplementary Table 1. The levels of all transcripts were normalized to levels of mouse or human actin transcript levels. QRT-PCR was performed using CFX96-Opus QPCR machine (BioRad, USA) and iQ-SYBR Green Supermix (BioRad, USA) or MAXIMA SYBR/R QPCR mix (Fisher Scientific, USA) as described (53, 68). In qRT-PCR reactions, the standard curve was generated using 10-fold serial dilutions of known quantities of respective fragments. Total RNA from RAW macrophage and HaCaT cells was used to generate cDNA which was the template for initial amplification of the genes. The qRT-PCR products were then run on 1.2% agarose gel, and the fragments were then excised, concentration was measured and sent for sequencing at Eurofins Genomics (USA). Standards were made with the purified fragments for each gene and diluted starting from 1 ng to 0.00001 ng.

Macrophages and HaCaT cells treated with either salivary gland lysates generated from unfed or fed ticks or with rGST or rGST-Otlip were collected and lysed in RIPA lysis buffer with protease inhibitor. Immunoblotting analysis was performed as described (68, 69). Total protein lysates (25 µg) from these cells were separated on 12% SDS-PAGE gels for IGFBP3, Bcl2 and beta-Actin (control) and 15% SDS-PAGE gel for Caspase-3 immunoblotting analysis. After gel electrophoresis, blots were blocked with 5% milk buffer and probed with IGFBP3 rabbit polyclonal antibody (cat. no. A16052, ABclonal, USA) or Caspase-3 p12 Rabbit monoclonal antibody (cat. no. A19664, ABclonal, USA) or Bcl2 Rabbit polyclonal antibody (cat. vo. A0208, ABclonal, USA) or beta-Actin rabbit monoclonal antibody (cat. no. 8457S, Cell Signaling Technologies, USA), followed by anti-rabbit HRP-conjugated secondary antibodies (cat. no. 7074S, Cell Signaling Technologies, USA). Antibody binding was detected with in-house ECL reagent and hydrogen peroxide (H₂O₂). ECL reagent was made in the lab based on the information given in the following link, https://bitesizebio.com/8970/how-to-make-your-own-ecl/ and the protocol was modified to make it user-friendly and to increase the shelf life of the prepared reagents. Briefly, 1 mL of 250 mM luminol (Sigma, USA) dissolved in DMSO and 0.44 mL of 90 mM p-Coumaric acid (Sigma, USA) dissolved in DMSO were mixed with 10 mL of 1 M Tris-Cl, pH:8.5. The ECL mixture was stored at -20 °C as 1 mL aliquots. This 1 mL aliquot was mixed with 7.5 mL deionized water and stored at -20 °C as 1 mL aliquots of ECL reagent A. Then 1 mL of ECL reagent A was mixed with ECL reagent B (4 µl of 3% H₂O₂) for chemiluminescence detection. Blots were imaged using Bio-Rad Chemidoc imaging system and processed using Image Lab software.

After 24-hours post-treatment, cells were visualized under the Cytation 7 imager for the detection of any changes in cellular morphology. Images were taken on several distinct focal planes for each treatment group.

For Live/Dead assay, RAW macrophage and HaCaT cells were plated in a 96 well plate as five replicates for each treatment. Cells were plated at 1e4 cells per well. The cells were treated with 5 μg/mL/well rGST or rGST-Otlip. After 24 hrs post-treatment, the Live/Dead assay was performed using LIVE/DEAD™ Cell Imaging kit, Invitrogen as described in our previous study (69). Briefly, 1 mL of green dye was added to and mixed with the red dye and 25 μl was added to each well. The wells were incubated for 30 mins at 37°C. The wells were then visualized with Cytation 7 imager (BioTek, USA). The images of cells from green channel (488 nm) indicated the live/viable cells, and the cells in red channel (594 nm) indicated the dead cells.

MTT assay was carried out as five replicates in a 96-well plate as described (69). The cells were seeded at a density of 3e4 per well containing 200 µl medium. After 24 hours of treatments with 5 μg/mL/well rGST or rGST-Otlip, 20 μl aliquots of MTT solution (5 mg/mL in 1x PBS) were added to each well. After three hours of incubation at 37°C and 5% CO2, the culture medium with MTT solution was removed, and formazan crystals were solubilized with 100 μl DMSO. The plates were then read on a microplate reader in CYTATION7 imaging system (BioTek, USA) at 570 nm and 690 nm wavelengths. Graphs were plotted by subtracting the absorbance value at 690 nm from the absorbance value at 570 nm.

The siRNA for silencing igfbp3 expression and scrambled siRNA (control) were obtained from Santa Cruz Biotechnology (USA). The catalog numbers for igfbp3 and control siRNAs are sc-39588 and sc-37007, respectively. The gene specific siRNA products from Santa Cruz Biotechnology (USA) usually have pools of three to five target-specific 19–25 nt siRNAs. As per the company (Santa Cruz Biotechnology, USA) note, the control siRNA consists of a scrambled sequence that will not lead to the degradation of any specific cellular transcript. Murine raw macrophage cells were seeded to a density of 1e5 cells per well in a 12 well plate in 10% FBS supplemented RPMI media. Cells were incubated overnight at 37°C. Transfections were carried out as per the manufacturer’s instructions (Santa Cruz Biotechnology siRNA transfection protocol). Briefly, for each transfection, 2 µl of solution A (siRNA duplex) was diluted in 100 µl siRNA transfection medium. Similarly, 6 µl (peak activity as per the company’s recommendation) of solution B (transfection reagent) was diluted in 100 µl transfection medium. In the next step, the siRNA duplex solution or solution A was pipetted directly to the diluted solution B and the mixture was incubated for 30 minutes at room temperature. This was done for both the scrambled-siRNA and igfbp3-siRNA. The cells were washed with 1 mL of 1X PBS. Six wells were transfected with scrambled siRNA, and another six wells were transfected with igfbp3 siRNA. For each transfection, 500 µl of solution A+B mixture was overlayed onto the washed cells and the cells were incubated with siRNA for 6 hours. After 6 hours, the cells were analyzed and we then added 1 mL of RPMI with 2X FBS without removal of the transfection mixture and the cells were incubated overnight at 37°C. Next day, the medium containing the siRNA was aspirated from each well and further washed with 1 mL of 1X PBS. Fresh culture medium was added to the wells and subsequently 5µg/mL/well purified rGST-Otlip was added and incubated overnight at 37°C. The following day, imaging was performed in Cytation-7 imaging system. The samples (six replicates) were collected in RNA lysis buffer for RNA extractions or RIPA buffer containing protease inhibitor for protein extractions.

The TUNEL assay was performed using Click-iT Plus TUNEL assay kit (ThermoFisher Scientific, USA) following the manufacturer’s instruction. Briefly, cells were plated at a confluence of 1e5 cells per well in a 12 well plate. Next day, these cells were treated with 5 µg/mL/well rGST or rGST-Otlip. The TUNEL assay was performed 24 hours post treatment. In the first step, cells were fixed with 4% PFA (paraformaldehyde) and incubated at room temperature for 15 minutes. In the next step, cells were permeabilized with 0.25% Triton-X-100 in 1X PBS and incubated at room temperature for 20 minutes. Following two washes with 1X PBS the TdT reaction was performed by addition of 100 μl TdT reaction buffer per well and incubated for 10 minutes at 37°C. After a subsequent PBS wash, the Click-iT Plus TUNEL reaction cocktail was added to each well and incubated at 37 degrees Celsius for 30 minutes. In the final step, the reaction cocktail was removed, and washes were performed with 1X PBS solution. Following the washing step, imaging was performed immediately using Cytation 7 imager (BioTek, USA). The Alexa Flour 594 picolyl azide dye was the fluorophore used for detection and imaging of apoptosis.

Statistical analysis was performed using GraphPad Prism 9 software. To compare two groups, Mann-Whitney U test was used. In the analysis, P<0.05 was considered significant. Horizontal lines in the scatter plots represent the median value. Wherever necessary, statistical test and P values used are reported.