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Supplementary Figure legends
Supplementary Figure 1
LIN28B expression correlated with stemness marker SOX2 in breast cancer
tissues and clinical progress of lung cancer patients.
(A) IHC analyses were used for LIN28B and SOX2 expression in 102 human breast
cancer specimens. Analysis is shown for percentages of specimens with either low or
high LIN28B expression relative to SOX2 level. The representative cases are shown
on the left. Scale bars: 200 μm; 20 μm (insets). (B) IHC analyses were used for
LIN28B expression in 150 human lung cancer specimens. Scale bars: 200 μm; 50 μm
(insets). The representative cases are shown on the left. Kaplan-Meier analysis of
LIN28B expression in survival of patients with lung cancer is shown on the right.
Supplementary Figure 2
ALDH+ cells have stemness propoties and high expression of LIN28B in human
breast cancer cells.
(A) ALDH+ cells were isolated from MDA-MB-231 cells by Flow Cytometry (B)
Western blots for LIN28B, SOX2, OCT4 and NANOG in either ALDH+ or ALDHcells. (C) Mamosphere formation of either ALDH+ or ALDH- MDA-MB-231 cells
was detected by microscopy. Scale bar: 200 μm. (D) Tumor incidence of ALDH-/
ALDH+ MDA-MB-231 cells in limiting dilutions. The representative picture of mice
which were injected with either ALDH- cells (left side of mouse) or ALDH+ cells
(right side of mouse) was shown on the left.
Supplementary Figure 3
Tumor microenvironment activates IKKβ and up-regulates stemness in human
breast cancer cells.
(A) Experiment description of cancer cells co-cultured with THP1 cells (stimulated to
M2 phenotype). (B) Percentage of ALDH+ cells in MDA-MB-231 cells and
CD44+CD24- cells in MCF-7 cells that respectively were co-cultured with or without
THP1 cells were detected by Flow Cytometry. (C) Mamosphere formation of
MDA-MB-231 cells that were co-cultured with or without THP1 cells was visualized
by microscope. Scale bar: 100 μm. (D) Western blots for p-IKKβ, IKKβ and LIN28B
in MDA-MB-231 cells and LIN28B in MCF-7 cells that were co-cultured with or
without THP1 cells. (E) Expression of CD44 in MCF-7 cells that were co-cultured
with or without THP1 cells by Flow Cytometry.
Supplementary Figure 4
LIN28B promotes the stemness of MCF-7 cells.
(A) Western blots for p-IKKβ, p-RELA, SOX2, OCT4, NANOG and LIN28B in
either CD44+CD24- or CD44-CD24+ cells of MCF-7 cells. (B) Mammosphere
frequency of LIN28B-MCF-7 cells was calculated and representative images were
visualized by microscope (below). (C) Percentage of CD44+CD24- cells of
LIN28B-MCF-7 cells was detected by Flow Cytometry. (D) Expression of LIN28B in
CD44high, CD44low, CD44medium and total cells of MCF-7 cells by Flow Cytometry. (E)
Expression of MYC, SOX2, OCT4 and NANOG in LIN28B-MCF-7 cells.
Supplementary Figure 5
LIN28B maintains the stemness of H1299 cells and promotes tumor growth of
H1299 cells in NOD/SCID mice.
(A) Western blots for LIN28B in siLIN28B or shLIN28B of MDA-MB-231 and
H1299 cells. (B) Percentage of ALDH+ cells of shLIN28B-H1299 cells was detected
by Flow Cytometry. (C) Primary mammosphere frequency of shLIN28B-H1299 cells
were calculated and representative single sphere images were visualized by
microscope (below). (D) Secondary mammosphere frequency of shLIN28B-H1299
cells was calculated and representative sphere images were visualized by microscope
(below). Scale bar: 200 μm. (E) Matrigel invasion assay for shLIN28B-H1299 cells
were detected by microscope and OD value. Scale bar: 100 μm. (F) The primary and
metastasis tumor of shLIN28B-MDA-MB-231 cells was monitored by PET scan. (G)
The curve of tumor growth in NOD/SCID mice after injected with shLIN28B-H1299
cells subcutaneously (n= 5/group). (H) Survival curve of tumor-bearing mice in
different groups. *P<0.05.
Supplementary Figure 6
IKKβ up regulates expression of LIN28B and sustains cancer stemness in human
lung cancer cells.
(A) Western blots for IKKβ in MDA-MB-231 and H1299 cells transfected with 4
different siIKKβ constructs respectively. (B) Western blots for IKKβ, RELA, RELB,
LIN28B, SOX2, TCF7L2 and β-catenin in MDA-MB-231 and H1299 cells
transfected with siIKKβ, siRELA or siRELB, respectively. (C) Western blots for
p-IKKβ, IKKβ, p-RELA, RELA, LIN28B, SOX2, OCT4 and NANOG in H1299 cells
treated by IMD-0354 at doses of 10, 50, 100 μM. (D) qPCR for LIN28B, SOX2,
OCT4 and NANOG in H1299 cells that were treated with IMD-0354 (10 μM). (E)
Percentage of ALDH+ cells of H1299 cells that were treated with IMD-0354 (5 μM)
was detected by Flow Cytometry. (F) Mamosphere frequency of H1299 cells that
were treated with IMD-0354 (2 μM) were calculated and representative images were
visualized by microscope (below). Scale bar: 200 μm.
Supplementary Figure 7
TCF7L2 is required for IKKβ mediated activation of LIN28B which regulates
Wnt/TCF7L2 signaling via direct binding to the mRNA of TCF7L2 in H1299
cells.
(A) Western blots for TCF7L2 and LIN28B in H1299 cells that were treated with
siTCF7L2. (B) Chromatin immunoprecipitation (ChIP) was performed to verify
TCF7L2 binding with LIN28B gene in H1299 cells. (C) Luciferase reporter system
was used to identify the TCF7L2 binding site of LIN28B gene in H1299 cells which
were transfected with plasmids containing both site 1/ site 2, site 1, site 2 or mutated
site 2 separately. (D) Reporter plasmid containing both site1/site2 and a β-catenin or
control plasmid were co-transfected into H1299 cells, and luciferase activity was
detected. (E) TOP-flash/FOP-flash and Renilla pRL-TK plasmid were co-transfected
into shLIN28B-H1299 cells, and luciferase reporter activity was detected. Error bars
represent mean ± SD from 3 independent experiments. ***p<0.001.
Supplementary Figure 8
IMD-0354 suppresses tumor growth and metastasis of MDA-MB-231 cells in
NOD/SCID mice.
(A) Tumor volume at week 7 after MDA-MB-231 cells injected into 4th mammary fat
pads of NOD/SCID mice (n= 6 /group). (B) Body weight during 7 weeks after
MDA-MB-231 cells injected into 4th mammary fat pads of NOD/SCID mice (n= 6
/group). (C) Primary bioluminescent images of pulmonary metastasis at week 7 were
shown (n= 6 /group).
Supplementary Figure 9
IMD-0354 but not Taxol inhibit CSCs and tumor metastasis of MDA-MB-231
cells in vitro and in vivo.
(A) Percentage of ALDH+ cells of MDA-MB-231 cells that were treated with
IMD-0354 (2 μM) alone, Taxol (5 nM) alone and IMD-0354 (2 μM) + Taxol (5 nM)
for 5 days was detected by Flow Cytometry. (B) H&E staining of lung metastasis
tissue specimens of NOD/SCID mice injected with MDA-MB-231 cells and
representative images were shown. Scale bars: 200 μm; 50 μm (insets). (C) Single cell
suspensions derived from MDA-MB-231 xenografts harvested on day 56 were
analyzed for ALDH activity using the ALDEFLUOR assay and representative images
were shown. (D) Primary bioluminescent images of pulmonary metastasis at week 7
were shown (n= 5 /group).
Supplementary Figure 10
IMD-0354 but not Taxol inhibit CSCs and tumor metastasis of 4T1 cells in vitro
and in vivo.
(A) Balb/c mice were injected with 4T1 cells into the 4th mammary fat pads. Once
tumors reached a volume of ≥100 mm (6 days after tumor cells injection), mice were
treated by DMSO (control), IMD-0354 alone (5 mg/kg/i.p., daily for 4 days with 4
days’ withdrawal and for another 4 days), Taxol alone (daily for 4 days with 4 days’
withdrawal and for another 4 days) or both IMD-0354 and Taxol (daily for 4 days
with 4 days’ withdrawal and for another 4 days) (n= 5 /group). (B) Progression of
tumor volumes during days 6–30 in mice treated with DMSO, IMD-0354, Taxol or
Taxol + IMD-0354 (n= 5 /group). (C) Lung weight at day 35 of mice treated with
DMSO, IMD-0354, Taxol or Taxol + IMD-0354 (n= 5 /group). (D) Single cell
suspensions derived from 4T1 xenografts harvested on day 35 were analyzed for
ALDH activity using the ALDEFLUOR assay and frequency of ALDH+ cells was
evaluated (n= 5 /group).