The human LEC HLE B-3 line were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in low-glucose DMEM (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology, Haimen, China) at 37°C in humidified atmosphere with 5% CO₂. Cells were seeded in 10-cm dishes and grown to 95% confluence. Following discarding the culture supernatant, cells were washed twice with PBS and lysed with cold radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100 and 1% protease inhibitor cocktail from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany] at 4°C for 30 min. Following centrifugation at 14,000 × g at 4°C for 20 min, the supernatant was harvested and kept as cell lysate. When 95% confluent, cells were washed twice with PBS and cultured in 5 ml low-glucose DMEM without FBS for 48 h. The cell supernatant was harvested and centrifuged at 3,000 × g at 4°C for 5 min to remove debris.

For hematoxylin and eosin (H&E) staining, HLE B-3 cells grown on glass slides were stained with hematoxylin and eosin (5 and 2 min, respectively, at room temperature), and examined under a light microscope (Olympus Corporation, Tokyo, Japan; magnification, ×400). For ICC of LMs, HLE B-3 cells grown on glass slides were incubated for 10 min at room temperature with 3% hydrogen peroxide, and blocked with 10% normal goat serum for 20 min at room temperature, followed by incubation for 60 min at room temperature with rabbit anti-LM antibody (1:200; cat. no. ab11575; Abcam, Cambridge, UK). Subsequently, samples were incubated for 30 min at room temperture with peroxidase conjugated anti-rabbit antibody (cat. no. PV-9000; OriGene Technologies, Inc., Beijing, China) without dilution and color development was performed with 3,3′-diaminobenzidine (DAB). Staining was visualized with a light microscope (Nikon TE300, Nikon Corporation, Tokyo, Japan; magnification, ×400), and images were captured with an attached digital camera and associated software (SPOT Basic™ image capture software; cat. no. SPOT53BE; SPOT Imaging, a division of Diagnostic Instruments, Inc., Sterling Heights, MI, USA).

When HLE B-3 cells reached 95% confluence, they were washed with PBS and cultured in fresh low-glucose DMEM medium with 5% FBS; this day was recorded as day 0. At days 0, 3, 6, 9 and 12, HLE B-3 BMs were harvested, as described previously (25,26). Briefly, HLE B-3 cells were washed with PBS four times, after which 0.03% ammonia and 0.1% Triton-PBS was added to remove HLE B-3 cells completely. Finally, HLE B-3 BM remained on the bottom of the dish, which was washed with double distilled water 4 times and harvested with 150 µl of 4X sample buffer [8% sodium dodecyl sulfate, 0.2 M Tris-Cl (pH 6.8), 40% glycerol, 0.2% bromophenol blue and 0.1 M dithiothreitol] at room temperature or 1 ml RIPA buffer on ice.

Human ALCs were obtained from autopsies from 3 individuals (1 female and 2 male) aged 25–79 years old from November to December of 2016 at the Eye Bank of Heilongjiang Province (Harbin, China). Human ALCs were obtained by a single ophthalmologist using central circular capsulorhexis method from anterior surface of transparent lens (27). All experiments were performed with the approval of the Internal Review Board of Harbin Medical University and were conducted in accordance with Declaration of Helsinki Principles. Informed consent was obtained from all patients prior to mortality or from their families following mortality for inclusion of autopsy data. Samples were harvested within 4–24 h of mortality. Human ALCs were dissected from the isolated transparent lenses and were lysed with cold RIPA buffer at 4°C overnight. Finally, the supernatant was kept as human ALC lysate following centrifugation at 14,000 × g at 4°C for 20 min.

Following careful dissection from transparent lenses, excised human ALCs were frozen at −80°C. Frozen human ALC tissues were transversely sectioned at 5-µm thickness, mounted on glass slides, fixed with cold acetone for 15 min at 4°C, followed by H&E staining or IHC with identical steps to ICC, as aforementioned.

Protein concentrations in the HLE B-3 cell lysate, HLE B-3 cell culture supernatant, human ALC lysate and various HLE B-3 BMs were measured using a Bicinchoninic Acid Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol.

The total LM levels were measured by using commercially available LM ELISA kit in accordance with the manufacturer's protocol (E-EL-H0128c; Elabscience, Houston, TX, USA). The antibodies used in this kit were polyclonal antibodies (pAbs) against all types of LMα, LMβ and LMγ subunits.

Goat pAbs against LMα5 and LMγ3 (1:200, cat nos. sc-16592 and sc-133178; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit pAbs against LMα4, LMβ3 and LMγ2 (1:1,000, cat nos. C13067, C13071 and C30224; Assay Biotechnology Company, San Francisco, California), rabbit pAbs against LMγ1 (1:1,000, cat nos. ab69632; Abcam, Cambridge, UK), mouse monoclonal antibodies (mAb) against LMα4, LMα3, LMα2, LMα1, LMβ2 and LMβ1 (1:200, cat nos. sc-130540, SC-13586, sc-55605, sc-74418, sc-133241 and sc-17763; Santa Cruz Biotechnology, Inc.) were used in the present study.

Five known LMα chains, three known LMβ chains and three known LMγ chains in HLE B-3 cell lysate, HLE B-3 cell culture supernatant and HLE B-3 BMs harvested at days 0, 3, 6, 9 and 12, and human ALC lysate were analyzed by western blotting, as described previously (28). Briefly, antigen sources inclding HLE B-3 cell lysate, culture supernatant or protein lysate of human ALCs were mixed with 2X sample buffer, boiled for 2 min and separated by SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 1 h at room temperature, membranes were incubated with the aforementioned primary antibodies diluted in solution 1 (Toyobo Life Science, Osaka, Japan) at 4°C overnight. Following washes with TBS-T, membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:5,000; cat no. 31437; Thermo Fisher Scientific, Inc.), HRP-conjugated rabbit anti-goat IgG (1:5,000; cat no. 31433; Thermo Fisher Scientific, Inc.) or HRP-conjugated goat anti-rabbit IgG (1:5,000; cat no. 31463; Thermo Fisher Scientific, Inc.) diluted in solution 2 (Toyobo Life Science) for 1 h at room temperature. Under identical experimental conditions, normal rabbit IgG, normal mouse IgG or normal goat IgG (all 1:200; cat nos. sc-2027, sc-2025 and sc-2028; Santa Cruz Biotechnology, Inc.) were used as isotype controls. Finally, the antibody-antigen complex was visualized using an Enhanced Chemiluminescent kit (Beyotime Institute of Biotechnology).

All subsequent procedures were performed at 4°C, unless otherwise stated. A total of 1 ml HLE B-3 cell culture supernatant was incubated overnight on a shaker with protein G agarose (Beyotime Institute of Biotechnology) and anti-LMα4 rabbit pAb or anti-LMγ1 rabbit pAb. The same concentrations of normal rabbit IgG were used as an isotype control for IP. Precipitates, collected by centrifugation at 3,000 × g at 4°C for 2 min, were washed three times with PBS containing 0.5% Triton X-100, and were eluted from protein G agarose by boiling with 4X sample buffer for 2 min. Proteins in the supernatant were separated by SDS-PAGE and detected by western blotting, as aforementioned.

To investigate whether HLE B-3 cells were positive for LMs, H&E staining and ICC for LMs were performed. As depicted in Fig. 1A and B, LMs were widely expressed in the cytoplasm of HLE B-3 cells. To investigate the expression of LMs in HLE B-3 cell lysate and culture supernatant, ELISA and western blot analysis was performed. ELISA for the presence of LMs generated positive results in HLE B-3 cell lysate and culture supernatant (Fig. 1C). By western blotting, all 11 LM subunits were probed in the HLE B-3 cell lysate and culture supernatant. The HLE B-3 cell lysate was positive for LMα5, LMα4, LMα3, LMα2, LMα1, LMβ3, LMβ2, LMβ1, LMγ2 and LMγ1 subunits, whereas HLE B-3 cell culture supernatant was positive for LMα4, LMα2, LMα1 and LMγ1 subunits (Fig. 1D). The 10-fold concentrated HLE B-3 cell culture supernatant was also used for detection of 11 LM subunits by western blotting, and the results were identical to those obtained using the non-concentrated supernatant (data not shown).

To investigate what type of LM trimers appeared in HLE B-3 cell culture supernatant, IP experiments with antibodies against LMα1-LMα5 for IP, and antibodies against LMβ1-LMβ3 and LMγ1-LMγ3 for western blotting were performed. The immunoprecipitate obtained with antibodies for LMα1, LMα2, LMα3 and LMα5 were negative in western blotting with antibodies for all LMβ and LMγ subunits (data not shown). The immunoprecipitate obtained with antibodies for LMα4 was positive in western blotting with antibodies for LMβ1 and LMγ1 (Fig. 2A), indicating the presence of the LM411 trimer in the HLE B-3 cell culture supernatant. To confirm this result, the immunoprecipitate obtained with antibodies for LMγ1 was also analyzed by western blotting using antibodies against LMα4 and LMβ1, which proved that LMγ1 immunoprecipitates contained LMα4 and LMβ1 (Fig. 2B). These results confirmed that the LM411 trimer existed in the HLE B-3 cell culture supernatant.

HLE B-3 BMs were harvested at days 0, 3, 6, 9 and 12, and were analyzed for the expression of LMs by ELISA and western blotting. In LM ELISA, total LM levels in HLE B-3 BMs were similar at days 0, 3 and 6, but decreased to 70% at days 9 and 12 (Fig. 3A). Western blotting of HLE B-3 BMs detected the presence of LMα4, LMα2, LMα1, LMβ3 and LMγ1 subunits (Fig. 3B). Expression levels of LMα4, LMα2 and LMα1 in HLE B-3 BMs were relatively higher at days 3 and 6. LMβ3 expression levels increased with culture time. LMγ1 expression levels fluctuated with time irregularly, with the lowest levels at day 12.

To investigate LM distribution in human ALCs, H&E staining and IHC for LMs was performed. As depicted in Fig. 4A and B, LMs were primarily distributed in the ALC layer, close to the lens epithelial cells. To evaluate LM expression levels in human ALC, extracts from 2 human ALCs were examined by ELISA and western blotting with antibodies against all 11 LM subunits. In LM ELISA, total LM levels in two human ALCs (N1 and N2 samples) differed by ~3-fold (Fig. 4C), which may have resulted from the differences on age or gender. In western blotting, the N1 sample was weakly positive for LMγ2 and negative for the remaining 10 LM subunits, whereas the N2 sample was positive for LMα4, LMγ2 and LMγ1 subunits and negative for the remaining eight LM subunits (Fig. 4D).