A human ovarian clear cell adenocarcinoma cell line, SKOV3 (ATCC, Rockville, MD, USA) was used as an in vitro model for epithelial ovarian cancer. Cells were cultured in DMEM-F12 media containing 10% v/v fetal calf serum, 2mM l-glutamine, and penicillin (100 U/ml)/streptomycin (100 µg/ml) (Thermo Fisher). Cells were grown at 37°C under 5% v/v CO₂ until 80–90% confluency was reached.

Human C1q was purified as published earlier (12). Briefly, freshly thawed human plasma was made 5 mM EDTA, centrifuged at 5,000 × g for 10 min, and any aggregated lipids were removed using Whatmann filter paper (GE Healthcare, UK). The plasma was then incubated with non-immune IgG-Sepharose (GE Healthcare, UK) for 2 h at room temperature. C1q bound IgG-Sepharose was washed extensively with 10 mM HEPES, 140 mM NaCl, 0.5 mM EDTA, and pH 7.0 before eluting C1q with CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer (100 mM CAPS, 1 M NaCl, 0.5 mM EDTA, pH 11). The eluted C1q fractions were then passed through a HiTrap Protein G column (GE Healthcare, UK) to remove IgG contaminants, followed by dialysis against 0.1 M HEPES buffer, pH 7.5.

The recombinant forms of the globular head regions of human C1q A (ghA), B (ghB), and C (ghC) chains were expressed as fusions to Escherichia coli maltose-binding protein (MBP) and purified, as reported previously (13). Expression constructs pKBM-A, pKBM-B, and pKBM-C were transformed into E. coli BL21 (Invitrogen) cells in the presence of ampicillin (100 µg/ml). The primary bacterial culture was grown overnight by inoculating a single colony in 25 ml of Luria-Bertani medium containing ampicillin. The bacterial culture was then grown in a 1 L batch until OD₆₀₀ 0.6 and then induced with 0.4 mM isopropyl β-d-thiogalactoside (IPTG) (Sigma-Aldrich, UK) for 3 h at 37°C on a shaker and centrifuged (5,000 × g, 4°C, 15 min). Subsequently, the cell pellet for each fusion protein was lysed in 50 ml lysis buffer (20 mM Tris–HCl, pH 8.0, 0.5 M NaCl, 0.2% v/v Tween 20, 1 mM EGTA pH 7.5, 1 mM EDTA pH 7.5, and 5% v/v glycerol) containing lysozyme (100 µg/ml, Sigma-Aldrich, UK) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, UK) at 4°C for 30 min. The resultant cell suspension was sonicated at 60 Hz for 30 s with an interval of 2 min each (12 cycles) and centrifuged (16,000 × g for 30 min). The supernatant was diluted 5-fold with buffer I (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.2% v/v Tween 20, 1 mM EDTA pH 7.5, and 5% v/v glycerol) and passed through an amylose resin column (50 ml; New England Biolabs). The column was then washed extensively with buffer I (150 ml), followed by buffer II (250 ml of buffer I without Tween 20) before eluting 1 ml fractions of fusion proteins with 100 ml buffer II containing 10 mM maltose. The peak fractions were then passed through Pierce™ High Capacity Endotoxin Removal Resin (Qiagen) to remove lipopolysaccharide. Endotoxin levels in the purified protein samples were analyzed using the QCL-1000 Limulus amebocyte lysate system (Lonza). The assay was linear over a range of 0.1–1.0 EU/ml (10 EU = 1 ng of endotoxin), and the amount of endotoxin levels was <4 pg/μg of the recombinant proteins.

SKOV3 cells (0.5 × 10⁵) were grown on coverslips and incubated with human C1q, ghA, ghB, or ghC (10 µg/ml) in a serum-free DMEM-F12 medium for 1 h for analyzing cell binding, 24 h for apoptosis induction, and 15 h for mammalian target of rapamycin (mTOR) activation analysis. For binding analysis, the coverslips were washed three times with PBS and then incubated with rabbit anti-human C1q polyclonal antibody (MRC Immunochemistry Unit, Oxford, 1:200) for C1q and rabbit anti-MBP polyclonal antibody (Thermo Fisher) for MBP fusions of the globular head modules. Coverslips were washed three times with PBS, and then incubated with Alexa Fluor^® 488 (1:500, Thermo Fisher) and Hoechst (1:10,000, Thermo Fisher) for immunofluorescence analysis. Apoptosis was analyzed via immunofluorescence using a FITC-Annexin V apoptosis detection kit with propidium iodide (PI) (Biolegend). After 24 h incubation with proteins, the coverslips were then incubated with Annexin V-binding buffer containing FITC Annexin V (1:200) and PI (1:200) for 15 min and washed twice with PBS before mounting on the slides to visualize under a HF14 Leica DM4000 microscope. For mTOR analysis, following the 15-h incubation with proteins, the coverslips were washed three times with PBS. The cells were fixed and permeabilized using ice-cold 100% methanol at −20°C for 10 min, followed by 1 h incubation with rabbit anti-human mTOR (1:500, Sigma), and then 1 h incubation with Alexa Fluor^® 488 (rabbit anti-human 1:500, Thermo Fisher) and Hoechst (1:10,000, Thermo Fisher) for immunofluorescence analysis. In order to detect caspase 3 activation, the protein-treated SKOV3 cells were fixed and permeabilized using ice-cold 100% methanol at −20°C for 10 min, followed by 1 h incubation with rabbit anti-human cleaved caspase 3 (1:500, Cell Signaling), and then 1 h incubation with secondary antibody (anti-rabbit) probed with CY3 (1:500, Thermo Fisher) and Hoechst (1:10,000, Thermo Fisher) for immunofluorescence analysis.

SKOV3 cells (0.1 × 10⁶) were grown in a 12-well plate and incubated with C1q, ghA, ghB, ghC, or MBP (10 µg/ml) along with an untreated control in serum-free DMEM-F12 for 24 h. Cells were detached using 5 mM EDTA, pH 8.0 and centrifuged at 1,200 × g for 5 min. The cell pellet was re-suspended in 1 ml DMEM-F12, and 10 µl of cell suspension was stained with 10 µl of Trypan blue (60%; Sigma-Aldrich, UK) for cell counting using hemocytometer. The viable cells, i.e., unstained cells, were counted in 5 different optical fields with a threshold value of 200 cells per field.

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Thermo Fisher) assay was performed by incubating SKOV3 cells (0.1 × 10⁵) in a 96-well microtiter plate with C1q, ghA, ghB, ghC, or MBP (10 µg/ml each) and an untreated control in serum-free DMEM-F12 medium for 24 h, followed by incubation with 50 µg/µl MTT (5 mg/ml stock) per well for 4 h at 37°C. Majority of the media was removed leaving behind 25 µl per well, which was mixed thoroughly with 50 µl of dimethyl sulfoxide (DMSO) and incubated for another 10 min at 37°C. The absorbance was read at 570 nm using a plate reader.

SKOV3 cells (0.1 × 10⁷) were incubated with C1q, ghA, ghB, or ghC (10 µg/ml), along with an untreated control, in a six-well plate for 24 h, followed by cell detachment using 5 mM EDTA and centrifugation at 1,200 × g for 5 min. FITC Annexin V apoptosis detection kit with PI (Biolegend) was used, as per the manufacturer’s instructions. Compensation parameters were acquired using unstained, untreated FITC stained, and untreated PI stained SKOV3 cells. SKOV3 cells (0.1 × 10⁴) were acquired for each experiment and compensated before plotting the acquired data. For protein-binding analysis, SKOV3 cells (0.5 × 10⁶) were incubated with C1q, ghA, ghB, and ghC (10 µg/ml) and BSA-treated cells as a negative control. Cells were incubated at 4°C for 1 h, followed by 1 h incubation with rabbit anti-human C1q polyclonal antibody (1:200) for C1q and rabbit anti-MBP (Thermo Fisher, 1:200) for globular head modules treated cells as well as their respective BSA-treated controls. The cells were then incubated with Alexa Fluor^® 488 (1:1,000, Thermo Fisher) for 1 h and cells were washed with PBS three times between each step. Compensation parameters were acquired using unstained, untreated Alexa Fluor^® 488-stained SKOV3 cells. SKOV3 cells (0.1 × 10⁴) were acquired using Novocyte Flow Cytometer for each experiment and compensated before plotting the acquired data.

SKOV3 cells (0.1 × 10⁷) were plated in a six-well plate (Nunc) and incubated with C1q, ghA, ghB, or ghC (10 µg/ml), along with an untreated control in serum-free DMEM-F12 medium for 12 and 24 h. The cells were lysed within the wells using lysis buffer (50 mM Tris–HCL, pH 7.5, 10% v/v glycerol, and 150 mM NaCl) over ice for 10 min, before being gently transferred to a pre-cooled microcentrifuge tube, and centrifuged for 15 min at 13,000 × g at 4°C. The cell lysate was mixed with the treatment buffer (50 mM Tris pH 6.8, 2% β-merceptoethanol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) in order to run on SDS-PAGE (12% w/v) for 90 min at 120 V. The SDS-PAGE separated proteins were then electrophoretically transferred onto a nitrocellulose membrane (Sigma) using transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol, pH 8.3) for 2 h at 320 mA, followed by blocking with 5% w/v dried milk powder (Sigma) in 100-ml PBS at room temperature for 2 h on a rotatory shaker. The membrane was washed with PBST (PBS + 0.05% Tween 20) three times, 10 min each, and incubated with primary anti-human C1q (polyclonal antibody), anti-caspase 9, anti-caspase 3, or anti-cleaved caspase 3 antibodies (1:1000 Cell Signaling Technology) at 4°C overnight. The membrane was washed with PBST (three times, 10 min each) before incubating with secondary anti-rabbit IgG HRP-conjugate (1:1,000; Promega) for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence (Thermo Fisher) in the Biorad ChemiDoc MP imaging system, or using 3,3-diaminobenzidine (DAB) substrate kit (Thermo Fisher).

SKOV3 cells (0.5 × 10⁶) were incubated with C1q, ghA, ghB, or ghC (10 µg/ml) for various time points, and the centrifuged cell pellets were stored at −80°C. GenElute Mammalian Total RNA Purification Kit (Sigma-Aldrich, UK) was used, as per the manufacturer’s instructions, to extract total RNA, followed by treatment with DNase I (Sigma-Aldrich, UK) to remove any DNA contaminants. The concentration and purity of total RNA were determined by measuring the absorbance at 260 nm and 260:280 nm ratio, respectively, using NanoDrop 2000/2000c (Thermo Fisher Scientific). Total RNA (2 µg) was used to synthesize cDNA using High Capacity RNA to cDNA Kit (Applied Biosystems).

Forward and reverse primer sequences (Table 1) were designed using the web-based Basic Local Alignment Search Tool and Primer-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The qPCR reactions were performed to measure the mRNA expression level of various target genes. Each reaction was conducted in triplicates and consisted of 5 µl Power SYBR Green MasterMix (Applied Biosystems), 75 nM of forward and reverse primers, and 500 ng cDNA, making up to a 10 µl final volume per well. Relative mRNA expression was determined by qPCR using the 7900HT Fast Real-Time PCR System (Applied Biosystems). Samples were initially incubated at 50°C and 95°C for 2 and 10 min, respectively, followed by amplification of the template for 40 cycles (each cycle involved 15 s at 95°C and 1 min at 60°C). The gene expression was normalized using the expression of human 18S rRNA as an endogenous control. The cycle threshold (Ct) mean value for each target gene was used to calculate the relative expression using the relative quantification (RQ) value and formula: RQ = 2^−ΔΔCt, which was compared with the relative expression of 18S.

GraphPad Prism 6.0 was used to make graphs, and the statistical analysis was carried out using an unpaired one-way ANOVA test. Significant values were considered based on *p < 0.05, *p < 0.01, and ***p < 0.001 between treated and untreated conditions. Error bars show the SD or SEM, as indicated in the figure legends.

The qualitative binding analysis of C1q and recombinant form of individual globular head (ghA, ghB, and ghC) modules (10 µg/ml) to SKOV3 cells using immunofluorescence microscopy revealed the membrane localization of these proteins following 1 h incubation at 4°C (Figure 1A). The nucleus, stained with Hoechst, showed a blue fluorescence (Figure 1A, panel A). The bound proteins localized on the cell membrane via green fluorescence; all the four proteins showed similar binding pattern (Figure 1A, panels B and D). FACS analysis revealed similar binding pattern, consistent with the observations made under the immunofluorescence microscopy. SKOV3 cells, treated with C1q and globular head modules, were over 90% positive in the FITC positive quadrant, as compared to the control protein, BSA (Figure 1B).

C1q and globular head modules (at 10 µg/ml concentration) were incubated with SKOV3 cells for 24 h. Trypan blue dye exclusion (Figure 2A) revealed a significant decrease in the cell viability following incubation with C1q (~50%), ghA (~50%), ghB (~40%), and ghC (~55%). MTT assay also confirmed the above mentioned observations (Figure 2B). Furthermore, the qualitative apoptosis analysis was performed using immunofluorescence microscopy. A significant proportion of SKOV3 cells, when treated with C1q proteins (10 µg/ml) for 24 h, stained positive for cell membrane integrity marker, Annexin V (conjugated to FITC), which binds to phosphatidylserine (PS) of the membrane of cells undergoing apoptosis (Figure 3, panel B). The nucleus was stained using Hoechst, which showed blue fluorescence (Figure 3, panel A). No FITC fluorescence was detected in the untreated cells, suggesting that the integrity of the cell membrane was intact, and hence, the cells were still viable (Figure 3).

FACS analysis revealed that the treatment of SKOV3 cells with C1q (10 µg/ml) for 24 h yielded approximately 58% (C1q, quadrant Q2-2; Figure 4) cells positive for both FITC (Annexin V marker) and PI (stains DNA) quadrant, significantly higher than 1.42% untreated control cells (UT, quadrant Q2-2; Figure 4), which represented a cell population undergoing late apoptosis. In addition, 16% of cells stained positive for FITC only (C1q, quadrant Q2-4; Figure 4), which represented an early apoptotic cell population.

Similar trends were also observed when SKOV3 cells were treated with ghA, ghB, and ghC; approximately 50% (ghA and ghC) and 40% (ghB) of cells had undergone apoptosis after 24 h treatment as seen in quadrant Q2-2 (ghA, ghB, and ghC in Figure 4). Approximately, 15–20% of early apoptotic cells stained positive for FITC alone (quadrant Q2-4; Figure 4) following treatment with each globular head module. In the untreated cells, approximately 90% of the cell population was both FITC and PI negative (UT, quadrant Q2-3; Figure 4). A shift in the florescence intensity in the treated SKOV3 cells, as compared to the untreated cells, further confirmed these observations (Figure 4).

The mRNA expression level of pro-inflammatory cytokine, TNF-α, was significantly upregulated at 12 h following C1q (log₁₀ 1.2-fold) and globular head modules; ghA (log₁₀ 0.4-fold), ghB (log₁₀ 1-fold), and ghC (log₁₀ 0.7-fold) treatment in comparison to an untreated control, as determined by qPCR (Figure 5A). NF-κB was also significantly upregulated (log₁₀ 0.7-fold) at 12 h, as anticipated, consistent with TNF-α upregulation (Figure 5B). Both TNF-α (not shown) and NF-κB mRNA expressions were downregulated by 24 h (Figure 5C), which could be attributed to the late apoptosis stage. In addition, TNF-α transcriptional upregulation occurred at an earlier time point for ghA, i.e., at 6 h as compared to the other treated samples (data not shown). These results prompted us to further investigate the mRNA expression of pro-apoptotic makers of intrinsic (Bax) and extrinsic (Fas) apoptosis pathways.

C1q- and globular head module-mediated apoptosis was further investigated by analyzing the expression of pro-apoptotic genes, Bax and Fas, via qPCR. Fas was transcriptionally upregulated in the C1q (log₁₀ 0.4-fold) treated SKOV3 cells, most significantly at 12 and 24 h relative to the untreated control cells (Figure 6A). The globular head module-treated cells showed minimal mRNA expression levels at 12 h (data not shown) and significant upregulation of Fas at 24 h (log₁₀ 0.4-fold) (Figure 6B). Similar trend of minimal mRNA expression at 12 h (data not shown) and significant upregulation at 24 h (Figure 6C) were observed for Bax mRNA expression in case of ghA, ghB, or ghC. The activation of apoptotic pathway causes the loss of cell membrane integrity, which was previously observed in the immunofluorescence microscopy at 24 h, consistent with the upregulation of mRNA expression of these genes. To further validate these results, the activation of Caspase 3 was investigated via western blot.

Apoptotic markers, caspase 3 and cleaved caspase 3, were detected in the SKOV3 cells, treated with C1q and globular head modules, in comparison with the untreated cells. The cleaved caspase 3 was detected at 17 kDa after 24 h of C1q, ghA, and ghB treatment in the western blot (Figure 7B). A reduction in the abundance of the full length at 32 kDa was seen after 24 h in comparison to 12 h (Figure 7A) β-actin (45 kDa) was used as a loading control for 12 h and 24 h (Figure 7C). In addition, the activation of caspase 3 was also seen by immunofluorescence in parallel to apoptosis staining for Annexin V-FITC at 24 h following treatment with C1q or globular head modules. Activated caspase 3 was clearly visible in the cytoplasm probed with Cy3, suggesting that the SKOV3 cells were in the early stages of apoptosis as the membrane was stained positively for Annexin V-FITC, in comparison to the control where no staining was detected (Figure 7D). The cleaved caspase 3 in the ghC treatment sample could not be detected via western blot; however, activated caspase 3 was detected via immunofluorescence.

In addition to the apoptosis markers, we also investigated the expression of mTOR signaling pathways since it is frequently detected in ovarian cancers (14). The mRNA expressions of mTOR, RICTOR, and RAPTOR (Figure 8A) were significantly downregulated (~log₁₀ 0.5-fold) at 6 h, thereby suggesting that the effects of C1q and globular head modules can compromise mTOR signaling within the first few hours of the treatment. Additionally, the presence of mTOR was detected abundantly in the cytoplasm (green) of the untreated cells, compared to SKOV3 cells treated with C1q and globular heads at 15 h time point via immunofluorescence microscopy (Figure 8B).