Figure Legends
Figure 1. The diabetic tubular injury of KK-Ay mice was
alleviated in Empa administrated mice.
(A)
The Schematic diagram of the
experimental procedure.
(B) The appearance of mice. (C) The morphology changes of kidney. (D)
Representative micrographs of HE-stained kidney sections. (E) Body
weight. (F) urinary NAG, (G) urinary NGAL, and (H) urinary microalbumin.
Results are presented as means ±
SDMs. *P < .05,
significantly different as indicated; # P < .05 significantly
different from C57 group;
Figure 2. The apoptosis
and mitochondrial fission in KK-Ay mice were alleviated in Empa group,
and the AMPK phosphorylation, SP1
and PGAM5 expression in diabetic kidney was reversed in Empa group. (A)
The expression of apoptosis,
mitochondrial fission and mitochondrial fusion related proteins. (B) The
TUNEL assay in kidney. (C) The changes of mitochondrial CYT and DRP1.
(D) Quantification of apoptosis
related protein expressing. (E)
Quantitative data of DRP1S637,
mito-DRP1 and DRP1S616 expression. (F) The expression of
phosphorylated AMPK, SP1 and
PGAM5. (G) Quantification of t-DRP1 and MFN2 expression. (H)
Quantitative data of phosphorylated AMPK, SP1 and PGAM5. Results are
presented as means ± SDMs. *P < .05, significantly different
as indicated; # P < .05 significantly different from C57
group;
Figure 3. The Empa alleviated the apoptosis and mitochondrial
fission, and recovered the mitochondrial membrane potential in HG
treated HK2 cells. (A) The expression of apoptosis, mitochondrial
fission and mitochondrial fusion related proteins in HK2 cells. (B) The
JC-1 assay in HK2 cells. (C) Changes of mitochondrial CYT and DRP1 in
HK2 cells. (D) Change of
mitochondria morphology detected by Mito-tracker. (E) Quantitative data
of BCL-2/BAX in HK2 cells. (F)
Quantification of cleaved-CASPASE 3 and mito-CYT in HK2 cells.
(G)
Quantitative data of t-DRP1 and MFN2 expression. (H) Quantitative data
of DRP1S637 and mito-DRP1 in HK2 cells. Results are presented as means ±
SDMs. *P < .05,
significantly different as indicated; # P < .05 significantly
different from NG group;
Figure 4. The Empa alleviated mitochondrial fission through the
AMPK-SP1-PGAM5 pathway. (A)
The expression of phosphorylated
AMPK, SP1 and PGAM5 in HK2 cells. (B) The effect of si-SP1 and si-PGAM5
on the DRP1S637 and AMPK
phosphorylation. (C) The effect of
AMPK activator and inhibitor on
the expression of DRP1S637, SP1 and PGAM5.
(D)
Quantification of SP1 and PGAM5
expression in HG treated HK2 cells that subjected to AMPK activator or
inhibitor. (E) Quantification of DRP1S637 and AMPK phosphorylation in HG
treated HK2 cells that subjected to AMPK activator or inhibitor. (F)
Quantitative data of expression in phosphorylated AMPK, SP1 and PGAM5 in
HK2 cells. (G) Quantification of SP1, PGAM5 and phosphorylated AMPK
expression in HG treated HK2 cells that subjected to si-SP1 or si-PGAM5.
(H) Quantification of DRP1S637 and t-DRP1 expression in HG treated HK2
cells that subjected to si-SP1 or si-PGAM5. Results are presented as
means ± SDMs. *P < .05, significantly different as indicated;
# P < .05 significantly
different from NG group (Figure D. E. F); # P < .05
significantly different from HG group (Figure G. H)
Figure 5. SP1 binds to the promotor of PGAM5 and increased its
transcription in HK2 cells. (A) The predicted binding sites to promoters
within the SP1 sequence. (B) Putative SP1 binding motifs (bold and
underlined) within the promoter of PGAM5. (C, D) Overexpression of SP1
increased the protein level of PGAM5. (E) Mutation of the PGAM5 promoter
at 539 nt (Mutant 2) decreased the activity of PGAM5 and deprived its
responsiveness to SP1 overexpression. (F) PCR-based ChIP assay confirmed
the binding of SP1 to the PGAM5 promoter. Results are presented as means
± SDMs. # P < .05 significantly different from Vector;
Figure 6. Proposed model for the signaling pathway by which
PGAM5 participates in the diabetic tubular injury via an AMPK dependent
pathway.