Figure 2.
SHMT2 enzymatic activity is essential for BL cell growth and viability. (A) Immunohistochemical staining of SHMT2 in lymphoma biopsies and lymph nodes from healthy individuals. Magnification is indicated with 20× and 63×, respectively. Scale bar for 100 µM is indicated. (B) Quantification of IHC with classification into negative, moderate, or strong expression. GC, germinal center; LN, lymph node; MZ, marginal zone. (C) Competitive growth assay of BL cell lines expressing a constitutive shRNA vector targeting SHMT2 (shSHMT2#1) or a nontargeting control shRNA (shCtrl) together with RFP in coculture with wild-type cells. Percentages of RFP-positive cells were measured at indicated times, and obtained values were normalized to the values of control shRNA expressing cells (mean ± standard error of the mean [SEM]; n = 3; P < .001 compared with nontargeting control shRNA transduced cells [two-way ANOVA]). (D) Cell cycle analyses of the BL cell lines BL60 and Ramos constitutively expressing an shRNA against SHMT2 (shSHMT2#1) or a nontargeting control shRNA (shCtrl) (mean ± SEM is shown; n = 4 to 5; ***P < .001 in Student t test). (E) Western blot analyses of cleaved PARP (cPARP), total PARP, and cleaved and total Caspase-3 in BL60 and Ramos cells constitutively expressing an shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtr). GAPDH or β-actin served as loading controls (n = 3-4). For quantification see supplemental Figure 2F. (F) Rescue experiment in which BL60 cells carrying a doxycycline-inducible shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtrl) with GFP as fluorescent reporter were transduced with a lentiviral vector coding for the fluorescent reporter BFP only (empty vector) or BFP and an shRNA-resistant version of SHMT2 (SHMT2res) or a catalytically inactive mutant thereof (SHMT2res-K280A). Percentages of GFP/BFP positive cells were determined over time by flow cytometry and normalized to the values of day 2 after doxycycline induction of shRNA expression. The diagram displays the values obtained at day 6 after induction of knockdown (mean ± SEM; n = 3). ***P < .001 in Student t test. Western blot validation for successful SHMT2 reconstitution is shown below. GAPDH served as loading control (n = 3; for empty vector/shCtrl vs shSHMT2 P < .001 in paired Student t test; for SHMT2res and SHMT2res-K280A, P = ns in paired Student t test; P = .51 and P = .37, respectively). (G) Tumor volume in NSG mice that were subcutaneously transplanted with BL60 carrying a doxycycline-inducible shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtrl). Tumor volumes were measured upon intraperitoneal doxycycline induction (once per day) of shRNAs (mean ± SEM; n = 5; P < .01 in two-way ANOVA; **P < .01 in Bonferroni posttest; ***P < .001 in Bonferroni posttest).

SHMT2 enzymatic activity is essential for BL cell growth and viability. (A) Immunohistochemical staining of SHMT2 in lymphoma biopsies and lymph nodes from healthy individuals. Magnification is indicated with 20× and 63×, respectively. Scale bar for 100 µM is indicated. (B) Quantification of IHC with classification into negative, moderate, or strong expression. GC, germinal center; LN, lymph node; MZ, marginal zone. (C) Competitive growth assay of BL cell lines expressing a constitutive shRNA vector targeting SHMT2 (shSHMT2#1) or a nontargeting control shRNA (shCtrl) together with RFP in coculture with wild-type cells. Percentages of RFP-positive cells were measured at indicated times, and obtained values were normalized to the values of control shRNA expressing cells (mean ± standard error of the mean [SEM]; n = 3; P < .001 compared with nontargeting control shRNA transduced cells [two-way ANOVA]). (D) Cell cycle analyses of the BL cell lines BL60 and Ramos constitutively expressing an shRNA against SHMT2 (shSHMT2#1) or a nontargeting control shRNA (shCtrl) (mean ± SEM is shown; n = 4 to 5; ***P < .001 in Student t test). (E) Western blot analyses of cleaved PARP (cPARP), total PARP, and cleaved and total Caspase-3 in BL60 and Ramos cells constitutively expressing an shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtr). GAPDH or β-actin served as loading controls (n = 3-4). For quantification see supplemental Figure 2F. (F) Rescue experiment in which BL60 cells carrying a doxycycline-inducible shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtrl) with GFP as fluorescent reporter were transduced with a lentiviral vector coding for the fluorescent reporter BFP only (empty vector) or BFP and an shRNA-resistant version of SHMT2 (SHMT2res) or a catalytically inactive mutant thereof (SHMT2res-K280A). Percentages of GFP/BFP positive cells were determined over time by flow cytometry and normalized to the values of day 2 after doxycycline induction of shRNA expression. The diagram displays the values obtained at day 6 after induction of knockdown (mean ± SEM; n = 3). ***P < .001 in Student t test. Western blot validation for successful SHMT2 reconstitution is shown below. GAPDH served as loading control (n = 3; for empty vector/shCtrl vs shSHMT2 P < .001 in paired Student t test; for SHMT2res and SHMT2res-K280A, P = ns in paired Student t test; P = .51 and P = .37, respectively). (G) Tumor volume in NSG mice that were subcutaneously transplanted with BL60 carrying a doxycycline-inducible shRNA against SHMT2 (shSHMT2#2) or a nontargeting control shRNA (shCtrl). Tumor volumes were measured upon intraperitoneal doxycycline induction (once per day) of shRNAs (mean ± SEM; n = 5; P < .01 in two-way ANOVA; **P < .01 in Bonferroni posttest; ***P < .001 in Bonferroni posttest).

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