Journal: EMBO Reports

Article Title: Arginine methylation-dependent METTL14-SMN interaction regulates RNA m 6 A homeostasis

doi: 10.1038/s44319-025-00590-7

Figure Lengend Snippet: ( A ) Sanger sequencing confirmation of Mettl14 arginine methylation deficient mouse model. In total, 13 arginine resides located at the C-terminal disordered region of Mettl14 were mutated to lysine residues. ( B ) The arginine methylation levels of Mettl14 in WT, heterozygous (WT/RK), and homozygous (RK/RK) mice were detected by IP-western blot using tissue lysates from mouse spleen and thymus. ( C ) The protein expression levels of several DNA repair genes, including BRCA1, ATRIP, and PALB2, were detected by western blot using Thymus tissues from WT, WT/RK, and RK/RK mice. ( D ) Mettl14 arginine methylation deficiency increases the population of primitive hematopoietic cells (LK cells). ** P = 0.0019. ( E ) Mettl14 arginine methylation deficiency increases the population of myeloid (Mac1 + Gr1 + ) cells in the BM, but not in the spleen. * P = 0.013. ( F ) Mettl14 arginine methylation deficiency decreases the B lymphoid (B220 + ) population in the BM, but not in the spleen. * P = 0.011. ( G ) Mettl14 arginine methylation deficiency decreases the T lymphoid (CD3 + ) population in the BM, but not in the spleen. Data from three independent replicates were analyzed by Student’s t test and shown as mean ± SD. ** P = 0.0097. .

Article Snippet: Rabbit anti-PALB2 antibody , Proteintech , 14340-1-AP.

Techniques: Sequencing, Methylation, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Validated Functional Analysis of PALB2 Missense Variants for Use in Clinical Variant Interpretation

doi: 10.1101/2020.08.27.270553

Figure Lengend Snippet: (A) The TLR assay, adapted from Certo et al. , utilizes a cell line with a stably integrated construct encoding mutGFP (a mutant version of the GFP cDNA that does not produce green fluorescent protein at baseline due to an engineered I-SceI nuclease target site) connected via the T2A linker to an out-of-frame mCherry cDNA. Position relative to reading frame is indicated in parentheses, where +1 leads to gene expression, while +3 is 2 base pairs (bp) out of reading frame. A DNA double-strand break in mutGFP is induced by transfection with a plasmid encoding IFP and I-SceI nuclease. A plasmid encoding BFP and a truncated GFP construct (truncGFP) provides a homologous donor sequence that can be used by the cell to repair the DNA double-strand break via the homology-directed repair (HR) pathway (left side). However, if the donor sequence is absent, or if HR is not intact (right side), the cell can undergo mutagenic non-homologous end-joining (NHEJ) thereby restoring mCherry expression if it results in a 2-bp frameshift (about one-third of events). (B) Flow cytometry plots show mean fluorescence intensity of live, single cells. Expression of IFP (indicating presence of the I-SceI plasmid) versus BFP (indicating presence of the truncGFP donor plasmid) is shown in the upper panels. Expression of GFP (indicating successful HR) versus mCherry (indicating NHEJ) is shown on the lower panels. Untreated 293T/TLR cells are negative for BFP, IFP, GFP, or mCherry. Cells transfected only with truncGFP donor plasmid are reflected by a BFP positive population, otherwise negative for IFP, GFP, or mCherry. Cells transfected with I-SceI nuclease are IFP positive, and some of the population also expresses mCherry, indicating DSB repair by NHEJ due to absence of the homologous donor template. Cells transfected with all components of the TLR assay exhibit presence of both truncGFP and I-SceI (double positive BFP/IFP population), with distinct populations positive for mCherry or GFP indicating DSB repair by NHEJ or HR, respectively). (C) Optimized timing of the TLR assay used for siRNA knock-down and re-expression of BRCA2 and PALB2 variants in 293T/TLR cells. Stably transfected 293T/TLR cells are reverse transfected with siRNA at time 0, followed by transient co-transfection of rescue plasmid (pDEST-FLAG), I-SceI nuclease (co-expressing IFP) and truncGFP donor construct (co-expressing BFP) 24 hours later. Flow cytometry is performed 72 hours after DNA transfection to detect DDR outcomes.

Article Snippet: Primary antibodies used were a mouse monoclonal anti-FLAG antibody (1:1,000; #F1804, Sigma-Aldrich), a polyclonal rabbit antibody against an epitope between residues 200-250 of PALB2 (1:2,000; #A301-247A, Bethyl Laboratories), and a rabbit monoclonal antibody against GAPDH (1:2,500; #AB9485, Abcam).

Techniques: Stable Transfection, Construct, Mutagenesis, Expressing, Transfection, Plasmid Preparation, Sequencing, Non-Homologous End Joining, Flow Cytometry, Fluorescence, Cotransfection

Journal: bioRxiv

Article Title: A Validated Functional Analysis of PALB2 Missense Variants for Use in Clinical Variant Interpretation

doi: 10.1101/2020.08.27.270553

Figure Lengend Snippet: Typical flow plots for one run of the TLR assay. Row 1 (left to right): Controls for siRNA/DNA transfection and batch effects. Row 2 (left to right): Transient rescue with PALB2 variants L35P (potentially pathogenic missense variant) , N497Mfs*64 (known pathogenic), and A712V (known benign).

Article Snippet: Primary antibodies used were a mouse monoclonal anti-FLAG antibody (1:1,000; #F1804, Sigma-Aldrich), a polyclonal rabbit antibody against an epitope between residues 200-250 of PALB2 (1:2,000; #A301-247A, Bethyl Laboratories), and a rabbit monoclonal antibody against GAPDH (1:2,500; #AB9485, Abcam).

Techniques: Transfection, Variant Assay

Journal: bioRxiv

Article Title: A Validated Functional Analysis of PALB2 Missense Variants for Use in Clinical Variant Interpretation

doi: 10.1101/2020.08.27.270553

Figure Lengend Snippet: (A) TLR assay results for the PALB2 experiments are summarized, demonstrating HR function after siPALB2 knockdown and rescue with plasmids containing PALB2 variants or controls. HR function is rescued by PALB2 wild-type cDNA and benign validation controls but is impaired with empty vector and pathogenic validation controls. Each rescue condition is depicted across the x-axis. The y-axis represents the LMM normalized HR/NHEJ ratio as the mean ± SEM of at least three independent experiments. For wild-type and empty vector controls, N=40. Thresholds for normal and abnormal functional impact are indicated by gray dashed lines. (B) Lollipop diagram of PALB2 protein variants tested in the TLR assay, colored by functional impact. Benign validation controls are shown in blue, pathogenic validation controls are shown in black. VUS are coded by functional impact, based on the thresholds set for “normal” (yellow) or “abnormal” (red) assay read-out. VUS with intermediate assay read-out are labeled in pink. Coiled-coil (CC), chromatin association motif (ChAM), and WD40 protein domains (WD1-7, seven blades of the WD beta propeller) are indicated by colored boxes.

Article Snippet: Primary antibodies used were a mouse monoclonal anti-FLAG antibody (1:1,000; #F1804, Sigma-Aldrich), a polyclonal rabbit antibody against an epitope between residues 200-250 of PALB2 (1:2,000; #A301-247A, Bethyl Laboratories), and a rabbit monoclonal antibody against GAPDH (1:2,500; #AB9485, Abcam).

Techniques: Plasmid Preparation, Functional Assay, Labeling

Journal: DNA Repair

Article Title: Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

doi: 10.1016/j.dnarep.2017.02.010

Figure Lengend Snippet: Summary of results following depletion of target DDR gene using siRNA in combination with PARG siRNA or PARG inhibitors.

Article Snippet: This membrane was immunoblotted with antibodies against Poly(ADP-ribose) (1:400, 10H Enzo Life Sciences), PARG (1:250, Abcam), PARP1 (1:1000, Santa Cruz), BRCA1 (1:500, Santa Cruz), BARD1 (1:500, H-300, Santa Cruz), PTEN (1:1000, Cell Signaling), PALB2 (1:500, Novus Biologicals), FAM175A (1:1000, Bethyl) and β-tubulin (1:2000, Sigma) each diluted in 5% milk and incubated at 4 °C overnight.

Techniques:

Journal: iScience

Article Title: A high-throughput approach to identify BRCA1-downregulating compounds to enhance PARP inhibitor sensitivity

doi: 10.1016/j.isci.2024.110180

Figure Lengend Snippet:

Article Snippet: PALB2 (E9R2W) , Cell Signaling Technology , cat#: 30253; RRID:AB_2895010.

Techniques: Recombinant, Transfection, Purification, Phospho-proteomics, Flow Cytometry, Imaging, Software

Journal: Cell reports

Article Title: The homologous recombination factors BRCA2 and PALB2 interplay with mismatch repair pathways to maintain centromere stability and cell viability.

doi: 10.1016/j.celrep.2025.115259

Figure Lengend Snippet: Figure 1. BRCA2, but not PALB2 or RAD51, suppresses HAC loss in dividing cells (A) Schematic of the HAC loss assay. (a)–(d) represent time points in (B) and (C). (B and C) Western blot showing BRCA2, RAD51, and PALB2 siRNA depletion in HT1080 alphoidtetO dGFP-HAC cells. Lamin A was used as a loading control. (D) Division index (DI) after siRNA treatment. The DI was determined from a single biological experiment (three technical replicates for Blast, siMisNeg, siBRCA2, siRAD51, and siPALB2 and one technical replicate for siSTK38). SD from three technical replicates. (E) Rate of HAC loss after siRNA treatment. siSTK38 was included as a positive control. Error bars represent SD of at least three independent experiments. Unpaired t test. Data from each experiment were normalized to siMisNeg. (F) Top: western blot showing siRNA-mediated protein depletion. siBRCA2 (13) and siBRCA2 (23) indicate 18 and 36 nM siRNA, respectively. Bottom: representative images of metaphase spread and FISH staining. Endogenous centromeres were detected with a CENP-B box probe (red in the merged image). HAC was detected with a tetO probe (green in the merged image, white arrow). The scale bar equals 10 mm. The inset scale bar equals 2 mm. (G) Quantification of the three independent FISH experiments shown in (F). Unpaired t test. Data from each experiment were normalized to siMisNeg. 50 metaphase spreads were analysed per conditions for 3 independent experiments. Error bars represent SD. ns represents no statistical significance; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRCA2 Millipore Cat#OP95; RRID:AB_213443 RAD51 Biogenes Custom (Originally reported at Yata et al., 201475; PMID 24835992, also used in Saayman et al.37) Cat#7946 PALB2 Biorbyt Cat#orb412704 Lamin A Sigma Aldrich Cat#L1293; RRID:AB_532254 Histone H3 (phospho S10) Millipore Cat#06-570; RRID:AB_310177 RAD51 Abcam Cat#ab176458; RRID:AB_2665405 CENP-A GeneTex Cat#GTX13939; RRID:AB_369391 CENP-C Millipore Cat#ABE1957; RRID:AB_3075449 CENP-B Bethyl Laboratories Cat#IHC-00064; RRID:AB_669682 CENP-B Santa Cruz Biotechnology Cat#sc-376283; RRID:AB_10988421 CENP-T Abcam Cat#ab220280; RRID:AB_2938694 ACA (anti-centromere) Antibodies Incorporated Cat#15-234-0001; RRID:AB_2687472 gH2A.X Millipore Cat#clone JBW301, 05-636; RRID:AB_309864 Cyclin B1 Santa Cruz Cat#sc-245; RRID:AB_627338 STK38 Abnova Cat#H00011329-M01; RRID:AB_540741 MLH1 Abcam Cat#Ab92312; RRID:AB_2049968 Alpha Tubulin Cell Signaling Technology Cat#3873S; RRID:AB_1904178 IgG Invitrogen Cat#02-6502; RRID:AB_2532951 Goat anti-Rabbit HRP Dako Cat#P0448; RRID:AB_2617138 Goat anti-Mouse HRP Dako Cat#P0447; RRID:AB_2617137 Goat anti-Mouse Alexa Fluor 647 Invitrogen Cat#A21235; RRID:AB_2535804 Goat anti-Rabbit Alexa Fluor 647 Invitrogen Cat#A21244; RRID:AB_2535812 Goat anti-Mouse Alexa Fluor 555 Invitrogen Cat#A21422; RRID:AB_141822 Goat anti-Rabbit Alexa Fluor 555 Invitrogen Cat#A21428; RRID:AB_141784 Goat anti-Rabbit Alexa Fluor 488 Invitrogen Cat#A11070; RRID:AB_142134 Goat anti-Mouse Alexa Fluor 488 Invitrogen Cat#A11017; RRID:AB_2534084 Anti-human Alexa Fluor 647 Jackson ImmunoResearch Cat#109-605-088; RRID:AB_2337887 Bacterial and virus strains TOP10 Invitrogen Cat#C404010 Chemicals, peptides, and recombinant proteins Penicillin-streptomycin solution Life Technologies Cat#15140122 Sodium bicarbonate solution Sigma Aldrich Cat#S8761 Blasticidin Invivogen Cat#ant-bl-1 G418 Sigma Aldrich Cat#G8168 Exonuclease III Promega Cat#M1811 Mitomycin C Abcam Cat#Ab120797 Lipofectamine LTX Thermo Fisher Scientific Cat#A12621 Lipofectamine RNAiMAX Thermo Fisher Scientific Cat#13778075 FISH blocking solution Roche Cat#11096176001 Indole-3-acetic acid (IAA) Sigma Aldrich Cat#I5148 (Continued on next page) Cell Reports 44, 115259, February 25, 2025 19

Techniques: Western Blot, Control, Positive Control, Staining

Journal: Cell reports

Article Title: The homologous recombination factors BRCA2 and PALB2 interplay with mismatch repair pathways to maintain centromere stability and cell viability.

doi: 10.1016/j.celrep.2025.115259

Figure Lengend Snippet: Figure 6. MLH1 depletion neutralizes aberrant centromere structures in BRCA2- and PALB2-depleted RPE1 cells (A) Western blot showing MLH1 expression in RPE1 MLH1 KO clones generated using Cas9-sgMLH1. Tubulin was used as a loading control. Clone 3 (bold) was used for further study. (B) Western blot showing 72 h of siRNA-mediated depletion of BRCA2 and PALB2 in RPE1 MLH1 KO cells. Lamin A was used as a loading control. (C) Representative images of colony growth in siRNA-depleted RPE1 parental cells and MLH1 KO cells. (D) The percentage of cell viability in RPE1 parental cells and MLH1 KO cells after 96 h of siRNA-induced BRCA2 or PALB2 depletion. Data represent an average of three independent experiments, and error bars represent SD. Two-tailed t test. (E) Schematic of siRNA depletion of RPE1 MLH1 KO cells for exo-FISH. (F) Representative images of exo-FISH of RPE1 MLH1 KO cells after 96 h of siRNA-mediated depletion of BRCA2 or PALB2. (G) exo-FISH of RPE1 MLH1 KO cells with or without siRNA-mediated depletion of BRCA2 or PALB2. Samples were normalized to the siMisNeg cenFISH or human satellite II+III FISH (HSatFISH) signal without ExoIII treatment. Each datapoint represents one nucleus, and large colored datapoints represent the av- erages of each independent repeat. n = 3. At least 25 cells were analyzed per condition per repeat. Error bars represent SEM. RM one-way ANOVA with Tukey’s multiple-comparisons test. ns represents no statistical significance; p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRCA2 Millipore Cat#OP95; RRID:AB_213443 RAD51 Biogenes Custom (Originally reported at Yata et al., 201475; PMID 24835992, also used in Saayman et al.37) Cat#7946 PALB2 Biorbyt Cat#orb412704 Lamin A Sigma Aldrich Cat#L1293; RRID:AB_532254 Histone H3 (phospho S10) Millipore Cat#06-570; RRID:AB_310177 RAD51 Abcam Cat#ab176458; RRID:AB_2665405 CENP-A GeneTex Cat#GTX13939; RRID:AB_369391 CENP-C Millipore Cat#ABE1957; RRID:AB_3075449 CENP-B Bethyl Laboratories Cat#IHC-00064; RRID:AB_669682 CENP-B Santa Cruz Biotechnology Cat#sc-376283; RRID:AB_10988421 CENP-T Abcam Cat#ab220280; RRID:AB_2938694 ACA (anti-centromere) Antibodies Incorporated Cat#15-234-0001; RRID:AB_2687472 gH2A.X Millipore Cat#clone JBW301, 05-636; RRID:AB_309864 Cyclin B1 Santa Cruz Cat#sc-245; RRID:AB_627338 STK38 Abnova Cat#H00011329-M01; RRID:AB_540741 MLH1 Abcam Cat#Ab92312; RRID:AB_2049968 Alpha Tubulin Cell Signaling Technology Cat#3873S; RRID:AB_1904178 IgG Invitrogen Cat#02-6502; RRID:AB_2532951 Goat anti-Rabbit HRP Dako Cat#P0448; RRID:AB_2617138 Goat anti-Mouse HRP Dako Cat#P0447; RRID:AB_2617137 Goat anti-Mouse Alexa Fluor 647 Invitrogen Cat#A21235; RRID:AB_2535804 Goat anti-Rabbit Alexa Fluor 647 Invitrogen Cat#A21244; RRID:AB_2535812 Goat anti-Mouse Alexa Fluor 555 Invitrogen Cat#A21422; RRID:AB_141822 Goat anti-Rabbit Alexa Fluor 555 Invitrogen Cat#A21428; RRID:AB_141784 Goat anti-Rabbit Alexa Fluor 488 Invitrogen Cat#A11070; RRID:AB_142134 Goat anti-Mouse Alexa Fluor 488 Invitrogen Cat#A11017; RRID:AB_2534084 Anti-human Alexa Fluor 647 Jackson ImmunoResearch Cat#109-605-088; RRID:AB_2337887 Bacterial and virus strains TOP10 Invitrogen Cat#C404010 Chemicals, peptides, and recombinant proteins Penicillin-streptomycin solution Life Technologies Cat#15140122 Sodium bicarbonate solution Sigma Aldrich Cat#S8761 Blasticidin Invivogen Cat#ant-bl-1 G418 Sigma Aldrich Cat#G8168 Exonuclease III Promega Cat#M1811 Mitomycin C Abcam Cat#Ab120797 Lipofectamine LTX Thermo Fisher Scientific Cat#A12621 Lipofectamine RNAiMAX Thermo Fisher Scientific Cat#13778075 FISH blocking solution Roche Cat#11096176001 Indole-3-acetic acid (IAA) Sigma Aldrich Cat#I5148 (Continued on next page) Cell Reports 44, 115259, February 25, 2025 19

Techniques: Western Blot, Expressing, Clone Assay, Generated, Control, Two Tailed Test

Journal: BMC cancer

Article Title: NUSAP1 promotes pancreatic ductal adenocarcinoma progression by drives the epithelial-mesenchymal transition and reduces AMPK phosphorylation.

doi: 10.1186/s12885-024-11842-5

Figure Lengend Snippet: Fig. 6 Decreasing NUSAP1 expression inhibits PDAC cell proliferation. a Relative expression levels of NUSAP1 among the PDAC cell lines. b mRNA expression levels of NUSAP1 in PANC-1 and CAPAN-1 cells were detected by reverse transcription quantitative PCR after transfection of si-NUSAP1. c The protein expression level of NUSAP1 decreased significantly after transfection of cells with si-NUSAP1. d The proliferation of PDAC cells was measured using CCK-8 assays. e Visualization of DNA replication by EdU incorporation. Cell nuclei stained red represent DNA replication. f A significant reduction in colony formation ability was observed after transfection with si-NUSAP1. g Apoptosis rates of the si-NUSAP1 cell lines. Student’s t test was used for comparisons between groups. All experiments were performed in triplicate. *P < 0.05. NC, negative control; si, small interfering RNA.

Article Snippet: The siRNA targeting NUSAP1 and scrambled negative control siRNA (si-NC) were purchased from Santa Cruz Biotechnology, Inc. (cat. nos. sc-93,396 and sc-37,007).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, CCK-8 Assay, Staining, Negative Control, Small Interfering RNA

Journal: BMC cancer

Article Title: NUSAP1 promotes pancreatic ductal adenocarcinoma progression by drives the epithelial-mesenchymal transition and reduces AMPK phosphorylation.

doi: 10.1186/s12885-024-11842-5

Figure Lengend Snippet: Fig. 7 Decreasing the expression of NUSAP1 inhibits the migration and invasion of PDAC cells. a Migration of PANC-1 cells in the si-NC and si-NUSAP1 groups according to Wound healing assays. b Migration of PANC-1 and CAPAN-1 cells in the si-NC and si-NUSAP1 groups according to Transwell assays. c Invasion of PANC-1 and CAPAN-1 cells in the si-NC and si-NUSAP1 groups according to Transwell assays. Student’s t test was used for comparisons between groups. All experiments were performed in triplicate. *P < 0.05. NC, negative control; si, small interfering RNA.

Article Snippet: The siRNA targeting NUSAP1 and scrambled negative control siRNA (si-NC) were purchased from Santa Cruz Biotechnology, Inc. (cat. nos. sc-93,396 and sc-37,007).

Techniques: Expressing, Migration, Negative Control, Small Interfering RNA

Journal: BMC cancer

Article Title: NUSAP1 promotes pancreatic ductal adenocarcinoma progression by drives the epithelial-mesenchymal transition and reduces AMPK phosphorylation.

doi: 10.1186/s12885-024-11842-5

Figure Lengend Snippet: Fig. 9 NUSAP1 promoted progression of PDAC in vivo. a We randomly divided the mice into the NC and the KD groups (n = 5), PANC-1 cells stably transfected with an empty vector or the NUSAP1 shRNA were subcutaneously inoculated in them respectively. Then we treated the mice as described in the Methods. b The tumor sizes were tested using Vernier calipers. Tumor growth curves were constructed based on the tumor volumes measured in the mice. c Quantification of the average weights of collected tumors from the above experiments. d The expression of NUSAP1, Ki-67, BAX and Cleaved caspase-3 were determined in tumor tissue sections from the xenografts using IHC (scale bar, 40 μm, n = 5). e The expression of NUSAP1, Ki-67, BAX, and Cleaved caspase-3 in tumor tissue between the NC and the KD groups, as determined by the IHC score. Student’s t test was used for comparisons between groups. *P < 0.05, **P < 0.01. NC, negative control; KD, knockdown

Article Snippet: The siRNA targeting NUSAP1 and scrambled negative control siRNA (si-NC) were purchased from Santa Cruz Biotechnology, Inc. (cat. nos. sc-93,396 and sc-37,007).

Techniques: In Vivo, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Construct, Expressing, Negative Control, Knockdown