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Supporting Information:
Fig. S1. Impact of SH3GL2 overexpression in breast cancer cells. (A) Lentivirus mediated
SH3GL2 overexpression in the breast cancer cells. (B-D) SH3GL2 overexpression
significantly reduced proliferation (p=0.0001-0.0004), anchorage independent growth
(p=0.0001-0.0016) and invasion (p=0.0003-0.0006) of the breast cancer cells. EV: Empty
vector; SH3GL2: SH3GL2 transduced. (E) Empty vector transduction did not impact SH3GL2
and MFN2 expression in the breast cancer cells compared to the naïve cells. EV: Empty vector;
GL2: SH3GL2 transduced. Beta-actin was used as a loading control. All experiments were
performed in duplicate and data presented as the mean ± SE of the duplicate experiments.
Fig S2. SH3GL2 overexpression influences expression of various key growth regulators.
(A) Introduction of SH3GL2 increased the expression level of CDH1 and PTEN in the breast
cancer cells. (B) SH3GL2 overexpression enhanced the expression level of ATG5 but not
LC3B except for LC3BI in MDA-MB-231 cells. EV: empty vector transduced; SH3GL2:
SH3GL2 transduced cells; β-actin was used as loading control. (C-D) Confocal imaging
showing SH3GL2 co-localization with MFN2 (C) and PINK1 (D) (arrows) in the SH3GL2
overexpressing MDA-MB-231 cells. DAPI was used for nuclear staining. DIC: Differential
interference contrast. Scale bar: 100 µm. Magnification X 200. (E-F) Bioinformatic analysis
predicted various amino acid residues for possible interplay between SH3GL2, MFN2 and
PINK1.
Fig. S3. Identification of the phosphorylation sites in SH3GL2. (A), Identification of
various serine, threonine and tyrosine phosphorylation sites in the SH3 and BAR domain of
SH3GL2 by bioinformatic analysis. (B) The phosphorylation potential was predicted to be
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higher in the serine and threonine residues by bioinformatic analysis. (C-D) Identification of
highly conserved serine and threonine phosphorylation sites in SH3GL2 among various
species.
Fig. S4. SH3GL2 depletion promotes progression of breast cancer cells. SH3GL2 silencing
resulted in decreased MFN2, PINK1 and CDH1 expression (A) and an enhanced
Mitochondria/cytoplasm ratio of CYTC (B). C: Control SiRNA treated cells; KD: SH3GL2
specific SiRNA treated cells. Beta-actin and IMMT were used as cytoplasmic and
mitochondria loading controls. (C-E) Increased proliferation (p=0.0001), invasion (p=0.0003)
and reduced O2- production (p=0.0001) by the SH3GL2 depleted cells. Scale bar: 50 µm.
Magnification X 200. (F) The empty vector transduction has negligible influence on SH3GL2
or MFN2 expression in the breast cancer cells. C: Control SiRNA treated cells; KD: SH3GL2
specific SiRNA treated cells. Beta-actin was used as a loading control.
Fig. S5. Impact of SH3GL2 overexpression in vivo. (A) High expression level of SH3GL2
(p=0.0002) and CDH1 (p=0.01) (arrowheads) in the SH3GL2 overexpressing MDA-MB-231
mammary implant compared to the empty vector treated group. Magnification X 200. (B)
Enhanced expression of CDH1 in the SH3GL2 overexpressing MDA-MB-231 xenografts from
multiple mice. (C) Enhanced expression of SH3GL2, MFN2 and PINK1 in mitochondria of the
SH3GL2 overexpressing MDA-MB-231 xenograft. Beta-actin and IMMT were used as
cytosolic and mitochondrial loading controls. M: Mouse; Scale bar: 50 µm.
Fig S6. Association between MFN2 and/or SH3GL2 loss and breast cancer progression.
(A-B) Loss of MFN2 was not associated with clinical stage (p=0.28) or histological grades
(p=0.99) of the breast cancer patients. (C-D) There was no association between the loss of both
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SH3GL2 and MFN2 with clinical stage (p=0.09) or histological grades (p=0.22). (E) High
expression of SH3GL2 and MFN2 in normal breast ductal epithelial tissues obtained from
cancer-free healthy women underwent mammoplasty. H&E: hematoxylin and eosin staining;
SH3GL2: IHC with SH3GL2 antibody; MFN2: IHC with MFN2 antibody. Magnification X
200. (F) Detection of SH3GL2 in mt obtained from cancer free normal breast epithelial tissues
underwent mammoplasty. Purified mt lysate prepared from HMLE and MCF-10A cells were
used as positive controls for SH3GL2 detection. IMMT and beta-actin were used as
mitochondria and cytoplasm loading controls respectively.
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