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Deep Red, a lysosome-specific NIR probe, showed good
HepG2 cells was verified using X-gal staining (Figure 5g).
agreement with images obtained with KSA01 and KSA02,
with Pearsonꢀs correlation coefficient (PCC) values of 0.87
and 0.90, respectively (Figure S11a). In order to confirm that
fluorescence intensity changes in the green and red channels
are associated with the pH values, the plasmid pLVX-Puro-
LacZ with the LacZ gene for E. coli b-gal was transfected into
human liver cancer cells (HepG2) to construct a LacZ-(+)
cell line with increased E. coli b-gal expression which was
clearly verified by a western blot assay (Figure 4c). E. coli b-
gal, unlike mammalian b-gal, is located in the cytoplasm
which has a neutral pH, as represented in Figure 4h.[4c]
Treating LacZ-(+) HepG2 cells with KSA01 and KSA02
resulted in significant fluorescence enhancements in both the
green and red channels (Figure 4a,b,d–g), but more so in the
red channel which corresponds to the predominating “basic
form”. Additionally, the distribution of extralysosomal exog-
enous E. coli b-gal was further confirmed by the poor overlap
between the fluorescence in the red channel of KSA01 and
KSA02 and that in the NIR channel of LysoTracker Deep
Red in LacZ-(+) HepG2 cells, with low PCC values of 0.53
and 0.39, respectively (Figure S11b). These results indicated
that our probes were able to detect both endogenous and
exogenous b-gal in living cells, and they could also report on
the microenvironmental pH of b-gal.
Then KSA01 and KSA02 were used to simultaneously
monitor the changes in SA-b-gal activity and lysosomal pH
during senescence. Human lung fibroblastic cells (MRC-5)
were cultured from passage 21 (P21) to passage 24 (P24) to
obtain replicative senescent cells at different senescent
stages,[17] which were previously verified using X-gal staining
(Figure 5b). MRC-5 cells (P21) incubated with KSA01
exhibited weak fluorescence in the green channel and
negligible fluorescence in the red channel (Figure 5a,c). With
increasing passage number (P22-P24), the fluorescence in-
tensity of the MRC-5 cells gradually increased in both
channels, with the intensity from the red channel increasing
more rapidly (Figure 5a,c). At the same time, the sum of the
fluorescence intensity (IRed + IGreen) and the ratio of fluores-
cence intensity (IRed/IGreen) were also amplified (Figure 5d,e),
indicating that during senescence the SA-b-gal content and
lysosomal pH both increased gradually in MRC-5 cells. In
order to confirm this result, we investigated the fluorescence
imaging of KSA02 in HL-7702 cells treated with doxorubicin
(DOX) which has been shown to induce senescence by
damaging DNA (Figure S12).[4c] A western blot assay was
preformed to confirm the protein overexpression of senes-
cence-associated human b-gal in DOX-treated HL-7702 cells
(Figure 6d–f). Compared to untreated HL-7702 cells, the
fluorescence signals in the green and red channels were
enhanced in the DOX-treated senescent HL-7702 cells after
staining with KSA02 (Figure S12). Furthermore, the fluores-
cence intensity observed for the red channel was greater than
that for the green channel.
Then HepG2 cells treated by XL413 for different length of
time or not treated with this inhibitor were imaged after the
addition of KSA02. The untreated cells displayed negligible
fluorescence in both the green and red channels, while the
fluorescence intensity gradually increased in both the chan-
nels for the XL413-treated cells over time (Figure 5 f,h).
Moreover, with increasing treatment time, the enhancement
of the green channel gradually deceased while that of the red
channel continued to increase, which could be quantified by
determining total fluorescence intensity (IRed + IGreen) and the
fluorescence intensity ratio (IRed/IGreen) (Figure 5i,j). Our data
indicated that the SA-b-gal level as well as the lysosomal pH
of cancer cells gradually increased during senescence. More-
over, the pH values of lysosomes in HepG2 cells with
senescence were measured by using a ratiometric lysosomal
pH dye (LysoSensor Yellow/Blue DND-160) (Figure S13a,b)
and the results further demonstrated that the pH values of
lysosomes increased with senescence. KSA01 was also used
for the same imaging experiments (Figure S14) and the results
are consistent with those for KSA02 (Figure 5 f). However,
under the same conditions, the fluorescence intensity ratio
(IRed/IGreen) for KSA01 was higher than that for KSA02, which
can be explained by the lower pKa value of KSAP1 than that
of KSAP2. For senescent HepG2 cells undergoing XL413
treatment for 3 days, KSA02 output in the red channel was
negligible resulting in a reduced IRed/IGreen ratio. In compar-
ison, KSA01 exhibited better performance than KSA02 with
an increased IRed/IGreen ratio and a detectable red fluorescence
signal. Similarly, these cancer cells treated with XL413 for 5–7
days were not severely senescent and still exhibited low
lysosomal pH, making KSA01 a better probe to study the
progression of senescence. However, for the more severely
senescent HL-7702 cells (healthy cells) treated with DOX and
exhibiting higher lysosomal pH, KSA02 was chosen instead of
KSA01 for imaging due to the significant red channel
emission of KSA01 resulting in an excessive IRed/IGreen ratio.
Therefore, in order to obtain appropriate imaging results, the
IRed/IGreen ratio should not be too low or too high. As such, we
propose that for the early stages of senescence when the
lysosomal pH is low, KSA01 should be used, but for later
stages with higher lysosomal pH, KSA02 is more suitable for
use. In addition, Hep3B cells were treated with LY3177833,
a senescence inducer similar to XL413,[18] to obtain additional
senescent model cells, which was then verified by using
a western blot assay (Figure S15i). Then both KSA01 and
KSA02 were applied to study the senescence of these cells
and the results were consistent with those of the senescent
HepG2 cells (Figure S15), which further confirmed our
statements.
Based on the above confocal imaging findings, it can be
seen that the images obtained by the probes in senescent cells
are significantly different from those in SKOV-3 cells. In
order to display this difference more intuitively, we compared
the images of senescent HL-7702 cells enriched by SA-b-gal
and SKOV-3 cells overexpressing ovarian cancer-associated
b-gal using KSA02, as shown in Figure 6a and Figure S12.
KSA02 mostly exhibited red fluorescence signal in senescent
HL-7702 cells, while it mainly produced a green fluorescence
Next, we treated senescent HepG2 cells with our probes
to evaluate changes in SA-b-gal levels and the lysosomal pH.
HepG2 cells were treated with XL413 which induces sen-
escence by inhibiting the function of the DNA replication
kinase CDC7.[18] The senescence state of XL413-treated
Angew. Chem. Int. Ed. 2021, 60, 10756 –10765
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