Journal: bioRxiv

Article Title: Ubiquitin-dependent degradation of p21 Waf1/Cip1 is mediated by AMBRA1 to limit DNA replication stress

doi: 10.1101/2025.07.28.666126

Figure Lengend Snippet: a , Replication fork speed from DAOY and ONS76 cells depleted or not for AMBRA1 (DAOY siSCR : n = 546; DAOY siAMBRA1 : n = 537 / ONS76 siSCR : n =576; ONS76 siAMBRA1 : n = 552; in three technical independent experiments). b , Replication fork length in DAOY and ONS76 cells depleted or not for AMBRA1 and treated without (CTRL) or with S1 nuclease (S1) for 30’ (DAOY siSCR CTRL: n = 1002; DAOY siSCR + S1: n = 1008; DAOY siAMBRA1 CTRL: n = 1002; DAOY siAMBRA1 + S1: n = 1005 / ONS76 siSCR CTRL: n =1010; ONS76 siSCR + S1: n = 623; ONS76 siAMBRA1 CTRL: n = 764; ONS76 siAMBRA1 + S1: n = 1010; in three technical independent experiments). c , ( Left panel ) Representative images of Replication Inter-Origin Distance (IOD) analysis and the associated quantification in DAOY and ( Middle panel ) U2OS ( Right panel ) cells depleted or not for AMBRA1 and treated without (CTRL) or with ATRi for 30’ (DAOY siSCR CTRL: n = 126; DAOY siSCR + ATRi: n = 229; DAOY siAMBRA1 CTRL: n = 148; DAOY siAMBRA1 + ATRi: n = 368 / U2OS siSCR CTRL: n =162; U2OS siSCR + ATRi: n = 296; U2OS siAMBRA1 CTRL: n = 177; U2OS siAMBRA1 + ATRi: n = 398; in three technical independent experiments). d , Replication fork length in U2OS cells treated as in 4c (U2OS siSCR + siSCR : n = 513; U2OS siSCR + siCDKN1A : n = 561; U2OS siSCR + siCDKN1B : n = 516; U2OS siSCR + siCDKN1A / B : n = 564; U2OS siAMBRA1 + siSCR : n =533; U2OS siAMBRA1 + siCDKN1A : n = 527; U2OS siAMBRA1 + siCDKN1B : n = 540; U2OS siAMBRA1 + siCDKN1A /B: n = 564; in three technical independent experiments). e, QIBC analysis of PLA reaction for p21 and PCNA in scramble or AMBRA1-depleted U2OS. Before fixation cells were either pre-extracted ( Right panel ) or not ( Left panel ), then PLA reaction for p21-PCNA was performed and lastly cells were stained for EdU and Hoechst (2000 cells are displayed per the condition of three independent replicates). f , Representative images of QIBC analysis of PLA reaction for PCNA and FEN1 in scramble or AMBRA1-depleted U2OS and DAOY cells with its corresponding jittered boxplot quantification (n=1000 per condition in 3 independent biological replicates). Scale bar = 10 um. g , Jittered boxplot quantification of QIBC analysis of γH2AX ( Left panel ) and phospho-RPS32(S33) ( Right panel ) foci in scramble or AMBRA1-depleted DAOY S-phase cells treated or not with FEN1 inhibitor (n=1000 per condition in 3 independent biological replicates). h , ( Left panel ) QIBC analysis of γH2AX foci along cell cycle in DAOY cells depleted for the indicated genes stained for EdU and Hoechst (2000 cells are displayed per condition). ( Right panel ), Jittered boxplot quantification of γH2AX foci (n=1000 per condition in 3 independent biological replicates). i , Barplot representing fold-change cell number in DAOY cell line depleted or not for AMBRA1, treated for 48 hrs with the indicated cytotoxic compounds (n=3). Data are presented as mean value ±SD for scatter plots while median and IQR is shown for boxplots. n refers to biological independent samples. Data were analyzed using One-way ANOVA ( a , b , c, d, f, g, h ).

Article Snippet: Primary antibodies used for immunoblot (IB), immunoprecipitation (IP) immunohistochemistry (IHC), Proximity-Ligation Assay (PLA) and immunofluorescence (IF) were: α-TUBULIN (GeneTex GTX628802 - IB 1:10000), β-actin (Sigma A2066 - IB 1:5000), Myc (Santa Cruz sc-40 – IB 1:1000, IP 500 ng), HSP90 (Santa Cruz sc13119 IB -1:10000), AMBRA1 (Millipore ABC131 - IB 1:1000), AMBRA1 (G-6) (Santa Cruz sc-398204 - IB 1:1000), AMBRA1 (Novus 26190002 - 1:100), cyclin D1 (Cell Signaling 2978 - IB 1:1000), cyclin D1 (Abcam 16663 - IF 1:300 / IP 1:100), cyclin D2 (Cell Signaling - 3741 IB 1:1000), cyclin D2 (Santa Cruz sc-452 - IF 1:50), cyclin D3 (Thermo Fisher Scientific - MA5-12717 WB 1:1000), cyclin E2 (Cell Signaling 4132 - IB 1:1000), p21 Waf1/Cip (Cell Signaling 2947 – IB 1:1000 / IF 1:100 / IHC 1:100, IP 250 ng), p27 Kip (Cell Signaling 3686 – IB 1:1000, IP 250 ng), p27 Kip (Santa Cruz sc-528 – 1:100), Ki67 (Abcam 15580 IF 1:200), HA (Y11) (Santa Cruz sc-805 - 1-500), CUL1-NEDD8 (Cell Signaling 4995 - IB 1:1000), PARP / cleaved-PARP (Abcam ab32138 - IB 1:1000), pRb 780 (Cell Signaling 8307 - IB 1:1000) pRb 807/811 (Cell Signaling 9308 - IB 1:1000), Cdt2 (Abcam ab72264 IB - 1:500), FEN1 (Novus NB100-150 PLA - 1:100), Rb (Cell Signaling 9313 IB - 1:1000), CDK4 (Cell Signaling 12790 – IB 1:1000), CDK4 (Santa Cruz sc-23896 – PLA 1:50), CDK2 (Cell Signaling 2546 – IB 1:1000), CDK2 (Santa Cruz sc-6248 – PLA 1:50), DDB1 (Novus NBP1-33061 – IB 1:1000), DDB1 (BD Biosciences 612488 – PLA 1:100), PCNA (Santa Cruz sc-56 – IB 1:1000), PCNA (Abcam ab18197 PLA – 1:100) Chk1 (Santa Cruz sc-8408 - IB 1:1000), Chk1(Ser345) (Cell Signaling 2341 - IB 1:1000), H2AX p-S139 (Merck Millipore 05-636 - IF 1:500), H2AX p-S139 (Abcam ab22551 - IB 1:500), CDK2 pT160 (Cell Signaling 2561 - IB 1:500), ATM (Abcam ab2618 – IB 1:1000), ATM pS1981 (Abcam ab81292 – IB 1:5000), p53 (Santa Cruz sc-126 – IB 1:500), UBIQUITIN (Santa Cruz sc-8017 - IB 1:500), SKP2 (Santa Cruz sc-7164 – IB 1:1000).

Techniques: Staining

Journal: Experimental Physiology

Article Title: Effect of prolonged voluntary wheel running on oxidative stress and defence mechanisms in cortex and hippocampus of healthy female rats

doi: 10.1113/EP092815

Figure Lengend Snippet: BER and neurotrophic factor. (a) Representative immunoblots of target proteins in whole tissue extract from cortex and hippocampus. Proteins probed for are shown on the left. OGG1, 8‐oxoguanine DNA glycosylase 1; APE1, apurinic/apyrimidinic endonuclease 1; FEN1, Flap endonuclease 1, POLB, DNA polymerase β; BDNF, brain‐derived neurotrophic factor. (b–j) All values are normalized to actin and set relative to the sedentary group mean. Bars represent means ± SD. n = 10 and 9 for sedentary and running group, respectively. Differences were evaluated by Student's unpaired t ‐test (b–e, g–j) with Welch's correction (f, k). BER, base excision repair.

Article Snippet: Primary antibodies utilized were as follows: rabbit anti‐BDNF (Abcam, Cambridge, UK, cat. no. ab108319, 1:1000, RRID: AB_10862052), rabbit anti‐APE1 (Thermo Fisher Scientific, cat. no. PA5‐29157, 1:000, RRID: AB_2546633), mouse anti‐APE1 (Novus Biologicals, Littleton, CO, USA, cat. no. NB‐100‐116, 1:1000, RRID: AB_10080558), rabbit anti‐POLB (Abcam, cat. no. ab175197, 1:1000), rabbit anti‐Flap structure‐specific endonuclease 1 (FEN1) (Novus Biologicals, cat. no. NBP1‐84697, 1:1000, RRID: AB_11036988), rabbit anti‐8‐oxoguanine DNA glycosylase 1 (OGG1) (Novus Biologicals, cat. no. NB100‐106, 1:500, RRID: AB_10104097), mouse anti‐superoxide dismutase 1 (SOD1) (Santa Cruz Biotechnology, Dallas, TX, USA, cat. no. sc‐101523, 1:500, RRID: AB_2191632), mouse anti‐superoxide dismutase 2 (SOD2) (Santa Cruz Biotechnology, cat. no. sc‐137254, 1:200, RRID: AB_2191808), mouse anti‐catalase (CAT) (Santa Cruz Biotechnology, cat. no. sc‐271803, 1:500, RRID: AB_10708550), mouse anti‐heat shock protein 60 (HSP60) (Santa Cruz Biotechnology, cat. no. sc‐271215, 1:1000, RRID: AB_10607973), and mouse anti‐β‐actin (Sigma‐Aldrich, St Louis, MO, USA, cat. no. A2228, 1:10,000, RRID: 476697).

Techniques: Western Blot, Derivative Assay

Journal: Experimental Physiology

Article Title: Effect of prolonged voluntary wheel running on oxidative stress and defence mechanisms in cortex and hippocampus of healthy female rats

doi: 10.1113/EP092815

Figure Lengend Snippet: Mitochondrial BER. (a) Representative immunoblots of target proteins in mitochondrial extracts from cortex and hippocampus. Proteins probed for are shown on the left. APE1, apurinic/apyrimidinic endonuclease 1; HSP60, heat shock protein 60. (b) Representative gel from in vitro AP endonuclease activity assay with mitochondrial extracts from hippocampus. S, substrate; P, product; AP, oligonucleotide with AP site; C, control oligonucleotide without a lesion. (c–f) All values are normalized to HSP60 (mitochondrial loading control) and set relative to the sedentary group mean. Bars represent means ± SD. Differences were evaluated by Student's unpaired t ‐test (c) or Mann–Whitney U ‐test (d–f). (c) n = 10 and 8 for sedentary and running group, respectively. (d) n = 10 and 7 for sedentary and running group, respectively. (e) n = 9 and 8 for sedentary and running group, respectively. (f) n = 7 and 9 for sedentary and running group, respectively. One outlier was excluded from the analysis in the sedentary group for (e, f). Other discrepancies in sample size are due to technical issues in assays or insufficient yield from mitochondrial purification. BER, base excision repair.

Article Snippet: Primary antibodies utilized were as follows: rabbit anti‐BDNF (Abcam, Cambridge, UK, cat. no. ab108319, 1:1000, RRID: AB_10862052), rabbit anti‐APE1 (Thermo Fisher Scientific, cat. no. PA5‐29157, 1:000, RRID: AB_2546633), mouse anti‐APE1 (Novus Biologicals, Littleton, CO, USA, cat. no. NB‐100‐116, 1:1000, RRID: AB_10080558), rabbit anti‐POLB (Abcam, cat. no. ab175197, 1:1000), rabbit anti‐Flap structure‐specific endonuclease 1 (FEN1) (Novus Biologicals, cat. no. NBP1‐84697, 1:1000, RRID: AB_11036988), rabbit anti‐8‐oxoguanine DNA glycosylase 1 (OGG1) (Novus Biologicals, cat. no. NB100‐106, 1:500, RRID: AB_10104097), mouse anti‐superoxide dismutase 1 (SOD1) (Santa Cruz Biotechnology, Dallas, TX, USA, cat. no. sc‐101523, 1:500, RRID: AB_2191632), mouse anti‐superoxide dismutase 2 (SOD2) (Santa Cruz Biotechnology, cat. no. sc‐137254, 1:200, RRID: AB_2191808), mouse anti‐catalase (CAT) (Santa Cruz Biotechnology, cat. no. sc‐271803, 1:500, RRID: AB_10708550), mouse anti‐heat shock protein 60 (HSP60) (Santa Cruz Biotechnology, cat. no. sc‐271215, 1:1000, RRID: AB_10607973), and mouse anti‐β‐actin (Sigma‐Aldrich, St Louis, MO, USA, cat. no. A2228, 1:10,000, RRID: 476697).

Techniques: Western Blot, In Vitro, Activity Assay, Control, MANN-WHITNEY, Purification

Journal: Cell reports

Article Title: Improved detection of DNA replication fork-associated proteins

doi: 10.1016/j.celrep.2024.114178

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-FEN1 antibody (H-300) , Santa Cruz , Cat# sc-13051; RRID: AB_2102786.

Techniques: Recombinant, Ubiquitin Proteomics, Mass Spectrometry, Software, Sonication, Fractionation