Additional file 1 of RETSAT associates with DDX39B to promote fork restarting and resistance to gemcitabine based chemotherapy in pancreatic ductal adenocarcinoma
Creators: name: Tu, Qiu
name: Liu, Xiuyun
name: Yao, Xiaoqing
name: Li, Ruixue
name: Liu, Gaojing
name: Jiang, Honglv
name: Li, Kaiqin
name: Chen, Qiongfang
name: Huang, Xiaoyan
name: Chang, Qing
name: Xu, Guoqiang
name: Zhu, Hong
name: Shi, Peng
name: Zhao, Bo
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Marine Biology
subject: Cancer

description: Additional file 1: Supplementary Fig. 1. RETSAT deletion sensitizes PDAC cells to gemcitabine. (A) The expression of RETSAT in KRAS mutant (n = 86) and KRAS wild type (n = 10) PDAC tumor tissues from TCGA database. (B, C) Immunostaining (B) and quantification (C) of cell proliferation marker Ki67 in parental and RETSAT-KO PANC-1 cells with or without gemcitabine treatment. (D, E) Immunostaining of cleaved caspase 3 (D) and flow cytometry based Annexin V apoptosis quantification (E) in parental and RETSAT-KO PANC-1 cells with or without gemcitabine treatment under 21% O2 or 0.3% O2. (F, G) Images (F) and quantification (G) of flow cytometry based Annexin V apoptosis of 3D culture PANC-1 spheroids under indicated treatments. (H, I) Images (H) and quantification (I) of in vivo bioluminescence of all mice at indicated time. (J-M) Bioluminescence quantifications of each group including parental with Vehicle (J), RETSAT-KO with Vehicle (K), parental with Gem (L), RETSAT-KO with Gem (M) were shown. Scale bar = 100 μm. n = 3 independent experiments unless otherwise stated. All data are presented as mean ± SEM. P values were calculated using a two-tailed student’s t test. Supplementary Fig. 2. RETSAT localizes onto DNA replication forks and has no effects on fork velocity or nascent DNA stability. (A) Immunofluorescence of RETSAT in PANC-1 cells with or without 0.2% Triton X-100 pre-wash ahead of paraformaldehyde fixation. (B) Co-immunostaining of RETSAT and telomeric PNA probe in BxPC-3 cells. (C, D) Co-immunostaining of RETSAT (green) and BrdU pulse labeled replication foci (red) in BxPC-3 and PANC-1 cells cultured under vehicle or HU induced stress conditions. (E) Co-immunostaining of RETSAT (green) and BrdU pulse labeled replication foci (red) in BxPC-3 cells under 21% or 0.3% O2 conditions. (F, G) Immunoblotting of RETSAT in PANC-1 cells treated with 4 mM HU (F) or 10 μM gemcitabine (G) at indicated time points. β actin was used as a loading control. (H, I) Experimental setup (H) and quantifications (I) of DNA fiber and neutral comet assay in PANC-1 cells treated with 50 nM gemcitabine at indicated time points. (J) Co-immunostaining of γH2A.X (green) and CIdU labeled fork restarting sites (red) in PANC-1 cells treated with 50 nM gemcitabine. (K) Quantification of fork velocity in parental and RETSAT-KO PANC-1 cells with or without HU treatment. At least 200 single forks were calculated in each sample. (L) Evaluation of nascent DNA stability in parental and RETSAT-KO PANC-1 cells with or without HU treatment, calculated by ratio of CIdU length divided by own IdU length. At least 200 single forks were calculated in each sample. Scale bar = 10 μm. n = 3 independent experiments unless otherwise stated. All data are presented as mean ± SEM. P values were calculated using a two-tailed student’s t test. Supplementary Fig. 3. Fork restarting system is crucial for PDAC cells resistant to gemcitabine. (A, B) Immunoblotting of BLM (A) and SMARCAL1 (B) in PANC-1/Gem-R cells transfected with shVector or indicated shRNA lentivirus. β actin was used as a loading control. (C) Quantification of fork restarting in PANC-1 and PANC-1/Gem-R cells with or without BLM and SMARCAL1 knocking down under vehicle or 50 nM gemcitabine treatment. (D, E) Images (D) and quantification (E) of flow cytometry based Annexin V apoptosis of PANC-1 and PANC-1/Gem-R with or without BLM and SMARCAL1 knocking down under vehicle or 10 μM gemcitabine treatment. n = 3 independent experiments unless otherwise stated. All data are presented as mean ± SEM. P values were calculated using a two-tailed student’s t test. Supplementary Fig. 4. Changes of replisome components in response to RETSAT knocking out using iPOND combined with LC-MS/MS identification. (A) Corresponding to Fig. 4A, schematic of iPOND assay combined with LC-MS/MS analysis. (B) Heatmap of absent proteins in RETSAT-KO PANC-1 cells compared with parental under vehicle treatment. (C) Heatmap of newly emerged proteins in RETSAT-KO PANC-1 cells compared with parental under vehicle treatment. (D) Heatmap of absent proteins in RETSAT-KO PANC-1 cells compared with parental under gemcitabine treatment. (E) Heatmap of newly emerged proteins in RETSAT-KO PANC-1 cells compared with parental under gemcitabine treatment. Supplementary Fig. 5. RETSAT interacts with DDX39B and avoids R-loop accumulation. (A) Co-immunostaining of γH2A.X (green) and R-loop (red) in PANC-1 cells with or without 10 μM gemcitabine treatment. S9.6 antibody was used to label R-loop. (B) Immunoblotting of RNase H1 in PANC-1 cells with or without RNase H1 ectopic expression. β actin was used as a loading control. (C, D) Immunostaining (C) and quantification (D) of γH2A.X (green) positive PANC-1 cells with or without RNase H1 ectopic expression under indicated treatment. (E) Immunoblotting of DDX39B in PANC-1/Gem-R cells with or without DDX39B knocking down. (F, G) Immunostaining (F) and quantification (G) of Ki67 in PANC-1/Gem-R cells with or without DDX39B knockdown under vehicle or 10 μM gemcitabine treatment. (H, I) Clone formation (H) and quantification (I) of PANC-1/Gem-R cells with or without DDX39B knocking down under vehicle or 10 μM gemcitabine treatment. (J) Flow cytometry based Annexin V apoptotic analysis of PANC-1/Gem-R cells with or without DDX39B knockdown under vehicle or 10uM gemcitabine treatment. (K) Co-immunoprecipitation using anti-RETSAT antibody combined with immunoblotting to confirm the interaction of RETSAT and DDX39B in PANC-1 cells under vehicle or gemcitabine treatments. GAPDH was used as a negative control. (L, M) Immunoblotting of RETSAT and DDX39B in parental, RETSAT-KO and DDX39B knocking down PANC-1 cells. β actin was used as a loading control. (N) Co-immunostaining of RETSAT (green) and BrdU pulse labeled replication foci (red) in PANC-1 cells with or without DDX39B under indicated conditions. (O) Immunoblotting of RNase H1 in RETSAT-KO PANC-1 cells with or without RNase H1 ectopic expression. β actin was used as a loading control. (P) Immunostaining of DDX39B in parental and RETSAT-KO PANC-1 cells with or without RNase H1 ectopic expression. Scale bar = 10 μm in (A) and (H), 50 μm in (C) and (F). n = 3 independent experiments unless otherwise stated. All data are presented as mean ± SEM. P values were calculated using a two-tailed student’s t test.