![Figure 2. MYC represses miR-150 expression in tFL. (A-B) The differences in MYC messenger RNA (mRNA) and miR-150 levels in B cells from transgenic iMycCα mice (MYC transgene controlled by the immunoglobulin [Ig] α heavy-chain enhancer) compared with wild-type (WT) mice. The splenic B cells were purified from MYC hemizygous and control (ctrl) spleens of relatively young mice before the development of any malignancy, which was verified by flow cytometry together with the sample purity (>95% B cells postpurification; supplemental Methods). The differences between groups were tested by unpaired t test. (C) miR-150 expression in WSU-NHL B-cell line transfected with siRNA against MYC (siMYC) or siRNA negative control (siNeg. Ctrl) (n = 4). Statistical differences were tested by paired t test. (D) Intensity of MYC IHC staining in paired FL-tFL samples from all 14 patients with available material (patient characteristics listed in supplemental Table 1 [FL patients 5-10 and 12-19]). Percentages of FL-tFL samples are shown, which were scored as MYC− (0% to 1% positive cells), mostly MYC− (1% to 5% positive cells), MYC+ (10% to 40% positive cells), strongly MYC+ (40% to 70% positive cells), and very strongly MYC+ (>70% positive cells). The number in each segment of the column indicates the number of samples with the corresponding intensity of IHC staining. Each arrow indicates the change in the IHC staining category in each of the analyzed pairs (note that the angle of the line does not show the trend of increase/decrease in percentage of positive cells). Representative images of the scoring of MYC staining in FL are shown in panel E and in supplemental Figure 7. The statistical differences between 13 paired FL-tFL samples were tested by Wilcoxon matched-pairs test. For 1 patient, only the material from the tFL sample was available for IHC (included in the graph but not in the paired statistical analysis). (E) Representative examples of differences in IHC staining for MYC between paired samples of FL (upper) and tFL (lower) from 3 patients. Original magnification ×400. (F) The miR-150 expression levels in FL samples scored as MYC− (0% to 1% positive cells), mostly MYC− (1% to 5% positive cells), weakly MYC+ (5% to 10% positive cells), or MYC+ (10% to 40% positive cells) for MYC staining. P values were assessed by unpaired t test. Representative images of the scoring of MYC staining in FL are shown in supplemental Figure 7 (MYC staining performed as tissue microarray blocks; supplemental Methods). (G) Immunoblot analysis of LIN28 protein (LIN28A and LIN28B) in B-cell non-Hodgkin lymphoma (B-NHL) cell lines and primary samples. The membrane was overexposed while detecting immunocomplexes, and no band(s) appeared in any B-NHL samples. Protein lysates from human embryonic stem cells (hESCs) were used as a positive control for LIN28A/B. *Two bands identified by the anti-LIN28B antibody represent two isoforms of LIN28B protein. (H) ChIP analysis for MYC binding in the region upstream of the MIR150 transcription start site in WSU-NHL cells. DNA from immunoprecipitates was assayed by qPCR on the independent amplicons upstream of the MIR150 gene (−12.7 to −0.35 kb relative to the MIR150 transcription start site [TSS]; supplemental Figure 12). The results are represented as percentage of DNA input; the error bars indicate the standard error of the mean. Enrichment quantitation of immunoprecipitates with control IgG antibody was calculated to equal 1, and MYC IP was normalized accordingly. NS, not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/132/22/10.1182_blood-2018-06-855502/5/blood855502f2.png?Expires=1735488317&Signature=k22uFksUFutGzTl1YC5wVc4GilLi09bIIUfojrQunLq0q3V6EerIMejQZdOvAjKwJMbTekOCgogwD29VnTtRPI-m-P6JaUamdWEBbV5CQhTeud642zEY8IvKB7vXLYhmGFjQyhn1C5NNaM7VwHmtKfcy9foT6vcLAZlX4dCtOnSwZ~8vtnGx9D-REhCrlv5vIXc8d1rHuVECfP5upHzvdhZmccGcQ2lbsf4BeKAYbSGEDkGUG5AKKsrVD-rEKKscen8d9ID4AfMmy~RyQ3OCIALcpu9mSId1tfyxfGSUiyEAbX27B2KVCJnof7eqPcOspU7KcF6S6gH7xQ6ECLXQ0g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MYC represses miR-150 expression in tFL. (A-B) The differences in MYC messenger RNA (mRNA) and miR-150 levels in B cells from transgenic iMycCα mice (MYC transgene controlled by the immunoglobulin [Ig] α heavy-chain enhancer) compared with wild-type (WT) mice. The splenic B cells were purified from MYC hemizygous and control (ctrl) spleens of relatively young mice before the development of any malignancy, which was verified by flow cytometry together with the sample purity (>95% B cells postpurification; supplemental Methods). The differences between groups were tested by unpaired t test. (C) miR-150 expression in WSU-NHL B-cell line transfected with siRNA against MYC (siMYC) or siRNA negative control (siNeg. Ctrl) (n = 4). Statistical differences were tested by paired t test. (D) Intensity of MYC IHC staining in paired FL-tFL samples from all 14 patients with available material (patient characteristics listed in supplemental Table 1 [FL patients 5-10 and 12-19]). Percentages of FL-tFL samples are shown, which were scored as MYC− (0% to 1% positive cells), mostly MYC− (1% to 5% positive cells), MYC+ (10% to 40% positive cells), strongly MYC+ (40% to 70% positive cells), and very strongly MYC+ (>70% positive cells). The number in each segment of the column indicates the number of samples with the corresponding intensity of IHC staining. Each arrow indicates the change in the IHC staining category in each of the analyzed pairs (note that the angle of the line does not show the trend of increase/decrease in percentage of positive cells). Representative images of the scoring of MYC staining in FL are shown in panel E and in supplemental Figure 7. The statistical differences between 13 paired FL-tFL samples were tested by Wilcoxon matched-pairs test. For 1 patient, only the material from the tFL sample was available for IHC (included in the graph but not in the paired statistical analysis). (E) Representative examples of differences in IHC staining for MYC between paired samples of FL (upper) and tFL (lower) from 3 patients. Original magnification ×400. (F) The miR-150 expression levels in FL samples scored as MYC− (0% to 1% positive cells), mostly MYC− (1% to 5% positive cells), weakly MYC+ (5% to 10% positive cells), or MYC+ (10% to 40% positive cells) for MYC staining. P values were assessed by unpaired t test. Representative images of the scoring of MYC staining in FL are shown in supplemental Figure 7 (MYC staining performed as tissue microarray blocks; supplemental Methods). (G) Immunoblot analysis of LIN28 protein (LIN28A and LIN28B) in B-cell non-Hodgkin lymphoma (B-NHL) cell lines and primary samples. The membrane was overexposed while detecting immunocomplexes, and no band(s) appeared in any B-NHL samples. Protein lysates from human embryonic stem cells (hESCs) were used as a positive control for LIN28A/B. *Two bands identified by the anti-LIN28B antibody represent two isoforms of LIN28B protein. (H) ChIP analysis for MYC binding in the region upstream of the MIR150 transcription start site in WSU-NHL cells. DNA from immunoprecipitates was assayed by qPCR on the independent amplicons upstream of the MIR150 gene (−12.7 to −0.35 kb relative to the MIR150 transcription start site [TSS]; supplemental Figure 12). The results are represented as percentage of DNA input; the error bars indicate the standard error of the mean. Enrichment quantitation of immunoprecipitates with control IgG antibody was calculated to equal 1, and MYC IP was normalized accordingly. NS, not significant.