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Chromatin remodeling factor LSH drives cancer progression by suppressing the
activity of fumarate hydratase
Xiaozheng He1,2,3,4,* , Bin Yan1,2,3,4,* , Shuang Liu1,*, Jiantao Jia1,2,3, Weiwei Lai1,2,3, Xing Xin1,2,3,4, Cene Tang1, Dixian Luo5, Tan Tan5, Yiqun Jiang1,2,3, Ying Shi1,2,3,4, Yating Liu1,2,3,4, Desheng Xiao6, Ling
Chen1,2,3,4, Shao Liu7, Chao Mao1,2,3, Gang Yin8, Yan Cheng9, Jia Fan10,11, Ya Cao1,2,3,4, Kathrin
Muegge12, Yongguang Tao1,2,3,4,+
Supplementary Figure Legends
Figure S1 Western blot analysis detected LSH expression a panel of NPC cells.
Equal amount of proteins from a panel of NPC cells was analyzed by Western
analysis using the antibody to LSH as indicated. β-actin was loading as housekeeping
protein.
Figure S2 Overexpression of LSH promotes cell growth, migration and invasion
in vitro.
MTT assay was applied to assess cell viability in HNE3 (A) cells that were
stably transfected with an LSH expression vector. LSH protein levels, as detected by
Western analysis, are shown in the inlet figure. The means and S.D. values are derived
from three independent experiments. * p <0.05. (B) Growth images in the plate
colony formation assay were measured in CNE1, HK1, and HNE3 cells that stably
overexpressed LSH. (C) The migration images of CNE1 and HNE3 cells were
analyzed with a modified Boyden chamber migration and invasion assay in the
absence or presence of LSH. (D) HNE3 cells with a stable expression of LSH were
analyzed for their ability to migrate in a wound healing assay at indicated time points.
CNE1 (E), HK1 (F) and HNE3 (G) cells stably expressing a control vector or LSH
were examined for the expression of ZO-1, E-cadherin, β-catenin and Vimentin by
Western analysis. (G) A representative experiments is shown for E-cadherin and
Vimentin in HK1 (H) and HNE3 (I) cells stably expressing a control vector or LSH
respectively using High Content Screening and High Content Analysis.
Figure S3 Overexpression of LSH promotes cancer progression in vivo.
(A) A xenograft model of tumor growth was established in nude mice to
evaluate the ability of LSH overexpression in HNE3 cells and MOCK HNE3 cells to
form tumors after injection with 3×106 cells for 63 days. (B) Tumor formation in nude
mice is shown after injection of HK1 cells stably expressing control vector or LSH
expression plasmids. (C) Metastasis was detected in the chest and abdomen (three out
of six mice) of CNE1-LSH injected mice and stained with H&E. (D) Tumors,
generated from HK1-MOCK and HK1-LSH groups, were examined for the
expression of LSH, ZO-1 and Vimentin by Western analysis. (E) H&E staining was
used for HK1-MOCK and HK1-LSH tumors in SCID mice and immunohistochemical
analysis was used for the detection of LSH, ZO-1 and Vimentin. The means and S.D.
values are derived from three independent experiments. ** p <0.01, *** p <0.001.
Figure S4. Overexpression of LSH promotes cancer progression in vivo.
Stable ectopic expression of LSH in HNE3 (A) and HK1 (B) cells did not
significantly affect body weight in nude mice. (C) H&E staining was used for HNE3MOCK and HNE3-LSH tumors in SCID mice and immunohistochemical analysis
was used to examine the level of LSH, ZO-1 and Vimentin in tissues from SCID nude
mice.
Figure S5 Knockdown of LSH inhibits cancer progression in vitro and in vivo.
Colony formation (A and B) was measured in C666-1 cells that had a stable
knockdown of LSH. A xenograft model of tumor growth was established in nude mice
to evaluate the ability of C666-1 cells with a stable knockdown of LSH to form
tumors with 1×106 cells for 27 days and tumor formation (C), tumor weight (D) and
body weight (E) was recorded. Data are represented as mean ± SD from seven mice
for each experimental group. C666-1 cells (F) and transplanted tumors (G) in nude
mice with a stable knockdown of LSH were examined for the expression of ZO-1, Ecadherin, β-catenin and Vimentin by Western analysis.
Figure S6 FH decreased FH expression.
(A) FH expression was analyzed in C666-1 cells after stable overexpression of
LSH. Western analysis (B) and immunohistochemical analysis (C) was used to
analyze the FH expression in transplanted tumor tissues after injection of CNE1-LSH
and HNE3-LSH cells. Western analysis (D) and immunohistochemical analysis (E)
was used to analyze the FH expression in transplanted tumor tissues after injection of
C666-1 cells with stable knockdown of LSH into nude mice. Relative FH/-actin
ratios were given. (F) Immunohistochemical analysis was used to examine FH protein
level in an NPC tissue array from NPC patients. FH was expressed at high levels in
nasopharyngeal tissues (NP), whereas it was expressed at lower levels in NPC tissues
(Tumor, Metastasis). (G) Summary of FH and LSH protein levels in 61 tissue samples.
Fisher’s test, two-tailed p<0.01.
Figure S7 LSH regulated TCA intermediates.
Relative concentrations of citrate (A) and α-KG (B) were measured in CNE1,
HK1, and HNE3 cells that stably overexpressed LSH. (C) Relative concentrations of
citrate and α-KG were measured in C666-1 cells after depletion of LSH. Ratio of αKG to succinate (D) and ratio of α-KG to fumarate (E) are shown in CNE1, HK1, and
HNE3 cells that stably overexpressed LSH. Ratio of α-KG to succinate and ratio of αKG to fumarate are shown in C666-1 after knockdown of LSH (F). The means and
S.D. values are derived from three independent experiments. * p <0.05, ** p <0.01,
*** p <0.001.
Figure S8. EBV status was not linked with TCA intermediates.
GC-MS was applied to analyze TCA intermediates in the serum of NPC
patients grouped according to the presence or absence of EBV.
Figure S9. TCA intermediates citrate and α-KG decreased other intermediates.
Relative concentrations of succinate, fumarate and malate were measured in
CNE1 (A) and HK1(B) cells. CNE1 cells were treated with citrate (C) and α-KG (D)
for one or three days as indicated and Western analysis performed to assess
expression of FH and EMT proteins.
Figure S10. TCA intermediates succinate, fumarate and malate affected the
protein levels of FH, EMT and IKKα.
CNE1 (A) and HK1 (B) cells were treated with succinate for one or three days as
indicated and Western analysis performed to assess expression of FH and EMT
proteins. CNE1 (C) and HK1 (D) cells were treated with fumarate for one or three
days as indicated and Western analysis performed to assess expression of FH and
EMT proteins. CNE1 (E) and HK1 (F) cells were treated with malate for one or three
days as indicated and Western analysis performed to assess expression of FH and
EMT proteins.
Figure S11. Western blot analysis detected FH expression a panel of NPC cells.
Equal amount of proteins from a panel of NPC cells was analyzed by Western
analysis using the antibody to FH as indicated. β-actin was loading as housekeeping
protein.
Figure S12. The concentrations of α-KG and citrate were analyzed in HNE3 cells
in the depletion of FH.
TCA intermediates α-KG and citrate were measured by GC-MS in HNE3 cells
after depletion of FH.
Figure S13 Western blot analysis detected EMT markers as indicated.
(A) Western analysis for detection of EMT markers as indicated in HK1 and
C666-1 cells. (B) Western analysis for detection of LSH protein in CNE1-LMP1 cells
and the parental cells.