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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.

A set of 14 HP variable refrigerant flow air conditioning system (VRF AC) with multi-compressor has been designed, and the output capacity control strategy of compressor(s) including the switching control model of standard compressor has been built. In the output capacity control model, a certain suction pressure is used as the pressure control target to adjust the output capacity of compressors, and a little pressure fluctuation is taken into account to amend the target pressure. Furthermore, in the compressor switching control model, the most favorable operation frequency region is determined on base of the energy efficiency characteristic and noise characteristic of the compressor. And, in order to solve the large fluctuation problem of the system running and frequent ON-OFF action of the standard compressor, the equal output capacity switching principle can be used to determine the thermo-on and thermo-off switched frequency points, and control the switching process of the compressor. Experiments demonstrate the feasibility of this control strategy to ensure the stability and reliability, improve the energy efficiency and reduce the compressor noise.