Compromised DNA repair is responsible for diabetes-associated fibrosis (2020)
Figure 5

Figure 5. Diabetes-Associated Persistent DNA Damage Signaling can be Reversed by Complimenting an Alternative Mode of DNA Repair

(A) Representative images of γH2AX positive nuclei in lungs of 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. The lungs were harvested 6-weeks after viral transduction. The empty vector served as control. Red represents γH2AX foci green cytoplasmic staining represents α-tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (from control lung) of the staining.

(B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in transduced lungs of 6-month STZ diabetic mice as described in Figure 5a (mean ± SEM; : p<0.01; N=8).

(C) Representative images of lungs from 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. Lungs were stained for cellular senescence-associated β-galactosidase as described in Figure 5a and visualized by bright field and polarized light, where the accumulated senescent areas are recognized by its blue staining (Scale 40µm).

(D) Quantitative analysis of persistent DNA damage associated inflammatory marker IL-6, in transduced lungs from 6-month STZ-diabetic mice, as determined by mean fluorescence intensity of respective group lungs (mean ± SD,: p<0.05, : p<0.001; N=8).

(E) Quantitative analysis of Masson's Trichrome stain for lungs of 6-month STZ induced diabetic mice with respective RAGE virions as described in Figure 5F (mean ± SD, : p<0.001; N=8).

(F) Representative images of Masson's Trichrome stain for lungs transduced with the respective RAGE expressing virions as described in Figure 5a and visualized by bright field and polarized light, where the accumulated ECM areas are recognized by its blue staining (Scale 40µm). The dotted lines represent the respective zoomed window.

(G) Pressure-volume curves of diabetic and non-diabetic mice were determined using the FlexiVent system 6 weeks after transduction using respective the RAGE virions, as described in Figure 5a. The curves represent group averages (N=8). The lungs studied in Figure 5a, b, c and d are identical (mean ± SD; Vector alone vs. RAGE-EE; : p<0.05; N=8).

Data information: Statistical significance was assessed by unpaired two-sided student's t-test. As stated, data represented as mean ± SD, : p<0.05;: p<0.01; : p<0.001.

Extended View Figure Legends

Legend Fig EV1

(A) Western blot analysis of pATM-S1981 indicating activation of DDR in post-natal lungs. Histone H1 was used as a loading control.

(B) Representative immunofluorescence images of post-natal lungs stained for the DNA damage marker 53BP1 (red) at the indicated days. Green cytoplasmic staining represents α-tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (shown P14) of the staining.

(C) Representative immunofluorescence images of post-natal lungs, stained for the proliferation marker phosphorylated-H3 Ser10 (pH3-S10; red) at the indicated days. Green cytoplasmic staining represents α-Tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (shown P14) of the staining.

(D) Mean percentage of the proliferation marker pH3S10 positive nuclei in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SD, ** P<0.01, N=8).

Legend Fig EV2

(A) Immunofluorescence analysis of DSBs associated foci, marked by γH2AX in lung adenocarcinoma (A549) cells, cultured in ribose (20mM) for 3 days and treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment (Scale 10µm). CML (marked in green) served as induction control. This experiment was performed simultaneously with the data shown in Figure 2A.

(B) Immunofluorescence analysis of DSBs associated foci, marked by γH2AX in lung adenocarcinoma (A549) cells, cultured in Fructose (40mM) for 3 days and treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment (Scale 10µm). CML (marked in green) served as induction control. This experiment was performed simultaneously with the data shown in Figure 2A and EV3A.

(C) Representative immunoblots from lysates of A549 cells cultured in the presence of the reducing sugars as indicated and treated with etoposide (5μM; upper panel) were probed for the DNA damage marker γH2AX. Histone H3 was used as a loading control.

Legend Fig EV3:

(A) Representative immunoblots from the lysates of A549 cells, cultured in the presence of reducing sugars as indicated, then treated with camptothecin (1μM; lower panel) and probed for the DNA damage marker γH2AX. Histone H3 used as a loading control.

(B) Immunofluorescence analysis of DSBs associated foci, marked by γH2AX, in lung adenocarcinoma (A549) cells, cultured in low glucose (5.5mM) for 5 days and then treated with camptothecin (1µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX, was monitored over 24 hours after drug treatment. CML (marked in green) served as induction control (Scale 10µm).

(C) Immunofluorescence analysis of DSBs-associated foci, marked by γH2AX in lung adenocarcinoma (A549) cells, cultured in high glucose (30mM) for 5 days and then treated with camptothecin (1µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX, was monitored over 24 hours after drug treatment. CML (marked in green) served as induction control (Scale 10µm).

(D) Immunofluorescence analysis of DSBs associated foci, marked by γH2AX in lung adenocarcinoma (A549) cells, cultured in ribose containing medium (20mM) for 3 days and then treated with camptothecin (1µM for 60 minutes). The resolution of DNA-DSBs foci, marked by γH2AX, was monitored over 24 hours after drug treatment. CML (marked in green) served as induction control (Scale 10µm).

(E) Immunofluorescence analysis of DSBs-associated foci, marked by γH2AX in lung adenocarcinoma (A549) cells, cultured in fructose containing medium (40mM) for 3 days and then treated with camptothecin (1µM for 60 minutes). The resolution of DNA-DSBs foci, marked by γH2AX, was monitored over 24 hours after drug treatment. CML (marked in green) served as induction control (Scale 10µm).

(F) Mean percentage of DSBs positive nuclei, marked by γH2AX, from the immunofluorescence analysis described in Supplementary Figure S3 d, e and f (more than 400 cells were analyzed for each bar). Cells cultured in glucose, fructose and ribose are more sensitive to camptothecin (doses as indicated) than low glucose (mean ± SD **: p<0.01 ***: p<0.001).

Legend Fig EV4:

(A) Representative immunoblots of lungs harvested from 4-months old +/db (non-diabetic lean control) or (db/db) diabetic mice and probed for γH2AX. Histone-H3 was used as a loading control.

(B) Representative images of lungs from 4-months old non-diabetic lean controls (+/db) and age-matched obese (db/db) diabetic mice, showing DNA damage foci, as marked by γH2AX (in red). Here tubulin was used as a morphology marker (shown in green); blue nuclear staining represents DAPI (Scale 10µm). The zoomed portions of each image were shown in thick white lines in the upper left corner.

(C) Representative immunoblots of kidneys harvested from 4-months +/db (non-diabetic control) or (db/db) diabetic mice and probed for γH2AX. Histone-H3 served as a loading control.

(D) Representative images of kidneys from 4-months old non-diabetic lean controls (+/db) and age-matched obese (db/db) diabetic mice, showing DNA damage foci, as marked by γH2AX (in red). Here tubulin was used as morphology marker (shown in green) and blue nuclear staining represents DAPI (Scale 10µm). The zoomed portions of each image were shown in thick white lines in the upper left corner of each image.

(E) Representative images of cellular senescence staining in lung and kidney of 4-months old lean controls (+/db) versus obese (db/db) diabetic mice. Sections stained for cellular senescence-associated β-galactosidase [SA-β-Gal] as described in Methods and visualized by bright field and polarized light, the senescent areas are recognized by its bluish-green staining (Scale 40µm). Eosin (pinkish-red) was used as a morphology stain.

Legend Fig EV5:

(A) Representative immunoblots showing the expression of RFP-RAGE, or RFP and γH2AX in transduced lungs of diabetic mice. Data presented in this Figure and in Figure 5 are from the same mice.

(B) Representative images of lungs from 6-month STZ diabetic mice transduced with the indicated AAV2/8 virions, as described in Methods. The lungs were harvested 6-weeks after viral transduction and anti-RFP was used for visualizing the expression of virions. Blue nuclear staining represents DAPI.

(C) Representative images of nuclei positive for the DNA-DSBs marker pATM in lungs from 6-month STZ-diabetic mice and control mice, transduced with the indicated AAV2/8 virions as described in Methods. The lungs were harvested 6 weeks after viral transduction. The empty vector expressing only RFP, served as control. Mean ± SD of 6 animals per group is shown. Green cytoplasmic staining represents α-tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (shown from RFP group) of the staining.

(D) Quantitative analysis of pATM positive nuclei in lungs of 6-month STZ induced diabetic mice with respective RAGE virions as described in Figure 4 (mean ± SD; **: p<0.01, (N=8).

(E) Quantitative analysis of the static compliance in lungs from 6-month STZ induced diabetic mice, transduced with AAV2/8 as described in Figure 4 (mean ± SD, *: p<0.05, N=8).

(F) Quantitative analysis of transduction effects of RAGE (AA or EE) or RFP pro-inflammatory, fibrotic and SASP gene expression in lung tissue. The mRNA of pro-inflammatory, fibrotic and SASP cytokines was significantly suppressed in RAGE-EE transduction group. The data were normalized against control value (mean ± SD, *: p<0.05, **: p<0.01).