Fig 1: RCR test.Blood samples taken from the patient implanted with LCAT-GMACs were tested for the presence of RCR using PCR amplification with env-specific primers. Total RNA from 400 μL of blood samples at the indicated time point was reverse-transcribed (50 ng) and subjected to PCR. The negative control contained no RNA, and the positive control contained 50 ng total RNA from SupT1 cells mixed with env-expressing GP + env-AM12 cells (105:1). PCR amplifications were performed using env-specific primers (labeled, env) or human actin-specific primers (labeled, actin). Gels were stained with ethidium bromide. Uncropped original image of 3 is shown as supplementary material (S).
Fig 2: In vivo tumorigenicity tests for LCAT-GMAC derived from the patient and control HeLa cells.Panel A. Graft cell volumes of NSG mice implanted with LCAT-GMACs or HeLa cells. LCAT-GMACs (1 × 107 cells/mouse, n = 10) or HeLa cells (1 × 105 cells/mouse, n = 5) were injected subcutaneously into NSG mice as described in Methods. The graft cell volumes of individual mice are shown in the upper panel. The mean graft cell volumes are shown in the lower panel. Due to overgrowth of the HeLa cells, one mouse was sacrificed 18 weeks after implantation, and two mice were sacrificed 20 weeks after implantation. The graft cell volumes of LCAT-GMACs implanted into the mice were unchanged, whereas HeLa cells developed a palpable mass by eight weeks after implantation. No graft cell volume was detected in mice injected with the cell suspension solution without cells (labeled Fibrin glue). Panel B. Histological findings in subcutaneous engraft of HeLa cells (the left image), cell suspension solution without cells (the middle image), and LCAT-GMACs from the patient (the right image). Representative images of histological analysis (hematoxylin and eosin staining) are shown. In NSG mice injected with HeLa cells, a robust outgrowth of the tumor was detected (indicated by asterisks). In contrast, no tumor was observed in mice injected with the cell suspension solution without cells (the middle image) or LCAT-GMACs (the right image). Note the fat cell accumulation in mice injected with LCAT-GMACs (indicated by arrow heads). Panel C. Western Blot Analysis of Immunoprecipitation (IP-Western) of sera of mice injected with LCAT-GMACs. Lane H denotes 1 μg of HDL; lane (+), 1 μg of HDL processed for immunoprecipitation; lane (−), none; lane PC, serum from HeLa cell injected mouse; lane NC, serum from a mouse injected the cell suspension solution without cells, lanes 1–10, serum from individual mouse implanted LCAT-GMACs. 100 μL serum samples (except for lane 2, 85μL) were immunoprecipitated and subjected to immunoblotting. The 60-kDa band detected in lane PC represents HeLa cell-derived LCAT protein, as revealed by IP western of the culture medium of HeLa cells. Uncropped original image of C is shown as supplementary material (S).
Fig 3: Changes in serum LCAT activity, serum lipids and lipoproteins.(A). Changes in serum LCAT activity before and after LCAT-GMAC implantation. Arrows labeled 10w, and 48w indicate the post-implantation 10 weeks and 48 weeks, respectively. (B). Serum levels of TG and TC. (C). Serum HDL-cholesterol and LDL-cholesterol levels. (D). Changes in serum free cholesterol (FC), cholesteryl ester (CE) level and %cholesteryl ester/cholesterol before and after the implantation. CE concentration was calculated according to Okazaki and Yamashita [26]: 1.684 × (TC-FC) in mg/dL. The dashed line indicates the basal level. (E). Polyacrylamide gel disc electrophoresis of serum lipoproteins. Electrophoretic separation of lipoprotein fractions was performed according to the procedure as described [24]. Bold arrows indicate IDL fractions.
Fig 4: Karyotyping and LAM-PCR analysis.(A and B) show representative karyotype images of LCAT-GMACs (A) and their parental cells (B), respectively. Metaphase cells (29 days of culture after fat harvest) were subjected to the Giemsa-banding technique, and processed chromosomes were captured and analyzed. LCAT-GMACs were analyzed for clonality using LAM-PCR (C and D). After retroviral transduction (day 7), cells were cultured for seven (labeled d14), eleven (labeled d18), 14 (labeled d21), 21 (labeled d28), 36 (labeled d43), and 43 (labeled d50) days and subjected to LAM-PCR. PC, positive control (human genomic DNA containing 0.1 mg/mL retrovirus vector DNA); NC, negative control with un-transduced parental cells from the patient. The ar shows non-specifically amplified bands with ≈100 bp that appear in all samples. Uncropped original images of C and D are shown as supplementary materials (CS and DS).
Fig 5: Changes in lipids in subclasses of lipoprotein separated by gel permeation-HPLC (GP-HPLC).(A). TG, CE, FC, and phospholipid (PL) levels in the 20 subfractions of lipoprotein separated by GP-HPLC are shown. Representative data in pre (−1) and-post-LCAT-GMAC implantation (+1 day, and 24–240 weeks) are shown. The values in all the time points are shown in Table 7. CE concentration was calculated according to Okazaki and Yamashita [26]. (B). Changes in TC, FC, and CE levels of the small HDL (subfraction 18) and very small HDL (subfraction 19) determined by GP-HPLC are shown. (C), Values of TC, FC, and CE contents are shown in the small HDL (subfraction 18) and very small HDL (subfraction 19) separated by GP-HPLC at the indicated time point.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for LCAT Antibody - BSA Free
Available conjugates: Available conjugates: UnconjugatedSizes Available: 0.1 ml (also 0.025 ml)