Fig 1.
LRRC33 and TGFβ1 mRNA expression in acute myeloid leukemia (AML) and other cancer cells.
Data is from Cancer Cell Line Encyclopedia database. (A) LRRC33 has the highest mRNA level in AML cells compared to other cancer cells. The p values are calculated by comparing mRNA level of AML cells to other cells through one-way analysis of variance (ANOVA) in Prism 7 Graphpad software. Error bars indicate the range of mRNA level of all the cell lines in each category. (B) LRRC33 and TGFβ1 mRNA expression are both high in AML cells. AML193 and MV4-11 are two representative cell lines studied in this research. Haemo/Lympo indicates other hematopoietic and lymphatic cells.
Fig 2.
Expression of LRRC33 and pro-TGF-β1 proteins in MV4-11 and AML193 cells by flow cytometry.
Cells were stained with anti-LRRC33 and anti-pro-TGFβ1 antibodies (Abs), and mouse IgG isotype control was used as primary antibody in control staining. BB515 or BV421-conjugated goat anti–mouse immunoglobulin G (IgG) was used as secondary Abs. Data are representative of three independent experiments. (A) Cell surface staining of LRRC33 and pro-TGF-β1 with or without PMA stimulation. The shaded curves indicate the IgG control. (B) Comparison of total LRRC33 and pro-TGF-β1 protein expression with or without PMA stimulation. Cells were permeabilized before staining. The amount of proteins is indicated by the mean fluorescence intensity (MFI). The background MFI from control IgG staining was subtracted from each group. Error bars represent the standard deviation of 3 replicates. The p values are calculated by 2- way ANOVA comparisons in Prism 7 Graphpad software. ns: not significant. *: p<0.1, **: p<0.01, ***: p<0.001, ****: p<0.0001.
Fig 3.
LRRC33 co-localizes with pro-TGF-β1 in MV4-11 and AML193 cells.
(A) Flow cytometry of double-staining of fixed and permeabilized AML193 and MV4-11 cells. Cells were stained with Alexa488-conjugated LRRC33 and APC-conjugated pro-TGF-β1 mAbs before and after PMA stimulation. Alexa488- and APC- directly conjugated non-specific IgG were used as control and the control staining are indicated in grey dots. Numbers of double positive cells are shown. (B) LRRC33 and pro-TGF-β1 co-localization visualized by confocal microscopy. Green: anti-LRRC33; Red: anti-pro-TGF-β1; Blue: DAPI staining.
Fig 4.
Detection of LRRC33-pro-TGF-b1 complex in MV4-11 and AML193 cells by Western blot (WB) and immunoprecipitation (IP).
(A) Cell lysates were analyzed by immune-blotting using biotinylated pro-TGF-β1 Ab. The LRRC33-pro-TGF-β1 complex band is indicated in non-reducing SDS-PAGE, and the band of pro-TGF-β1 alone is indicated in both non-reducing and reducing SDS-PAGE. (B) Co-immunoprecipitation. Cell lysates were subjected to immunoprecipitation using LRRC33 antibody or control mouse IgG as indicated, and the immunocomplexes were analyzed by WB using pro-domain antibody. The immunoprecipitated LRRC33-pro-TGF-β1 complex is indicated in non-reducing SDS-PAGE, and the pro-TGF-β1 pull down is indicated in the reducing SDS-PAGE.
Fig 5.
TGF-β1 activation in MV4-11 and AML193 cells assessed in TMLC-luciferase reporting system.
The luciferase activity is expressed in relative light units (RLU). Data are presented as the mean and standard deviation of four replicates and is the representative of three independent experiments on different days. (A) MV4-11and (B) AML193 cells, without or with PMA stimulation. (C) For MV4-11 and AML193 cells with PMA stimulation, incubation with 2.5 μg/ml anti-αv integrin mouse mAb 272-17E6 results in decrease of TGF-β1 activation. Non-specific mouse IgG is used as negative control. The p values are calculated by 2- way ANOVA comparisons in Prism 7 Graphpad software. ns: not significant. *: p<0.1, **: p<0.01, ***: p<0.001, ****: p<0.0001.