trim28 kap1  (Proteintech)

Journal: bioRxiv

Article Title: Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

doi: 10.64898/2026.06.01.729344

Figure Lengend Snippet: (A) Western blot time-course of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS cells after siRNA mediated knockdown of non-targeting control (NTG), UBR5, PPM1D , or UBR5 and PPM1D and exposure to 0 or 2.5 Gy irradiation. (B) Western blot of PPM1D, p-KAP1, KAP1, and UBR5 in U2OS isogenic cell lines after siRNA mediated knockdown of NTG or UBR5 and exposure to 0 or 2.5 Gy irradiation. (C) Immunofluorescence analysis of p-KAP1 in U2OS parental and isogenic cell lines transfected with NTG or UBR5 siRNA and exposed to 0 or 2.5 Gy irradiation. Representative images are shown on the left, and quantification of the mean fluorescence intensity (MFI) of p-KAP1 is shown on the right. n>230 cells for each condition were analyzed using CellProfiler. (D) Schematic of homology repair (HR) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (E) Schematic of non-homologous end joining (NHEJ) reporter (left) and normalized quantification (right) of GFP + U2OS parental or fl-PPM1D-only isogenic cells following siRNA mediated knockdown of NTG or UBR5 . (F) Representative images of COMET assay performed on U2OS parental or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA, 6 hours after treatment with 0 or 2.5 Gy irradiation. (G) Quantification of tail moments in COMET assay described in (F). n>160 comets for each condition were scored using OpenComet. (H) Schematic of cell growth competition assay. U2OS parental, control, or fl-PPM1D-only isogenic cells transfected with NTG or UBR5 siRNA were fluorescently labeled with BFP or RFP, the cells were mixed, exposed to 2.5 Gy irradiation then grown for 72 hours prior to quantification of the RFP:BFP ratio. (I) Normalized quantification of RFP to BFP ratio in the competition assay described in (H). (J) UBR5 truncating mutation frequency in PPM1D truncating mutation absent or present cell lines or primary tumors samples obtained from Depmap or TCGA. (K) UBR5 amplification frequency in PPM1D amplification or truncating mutation patient samples obtained from TCGA. Data are presented as mean ± SEM, n = 4 biological replicates for Figures D and E, and n = 3 biological replicates for Figure I. For Figures D and E, each data point indicates the normalized mean of three internal biological replicates from each independent test. Statistical significance was determined using unpaired two-tailed Student’s t-test for Figures C, D, E, G, and I; using Fisher’s exact test for Figures J and K; ns indicates non- significant ( p >0.05), * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: The following antibodies for immunoblotting were used in this study: PPM1D (Cell Signaling Technology, 11901), PPM1D (Santa Cruz Biotechnology, sc-376257), GAPDH (Cell Signaling Technology, 2118), Ubiquitin (Santa Cruz Biotechnology, sc-8017), LC3B (Cell Signaling Technology, 2775), COX IV (Cell Signaling Technology, 4844), HA (Cell Signaling Technology, 3724), V5 (Abcam, ab309485), Myc (Cell Signaling Technology, 2272), PSMA2 (Cell Signaling Technology, 2455), UBR5 (Cell Signaling Technology, 65344), p-KAP1 (S824) (Bethyl Laboratories, A300-767A), KAP1 (Cell Signaling Technology, 4124), K11-linked ubiquitin (kindly provided by Dr. Eugene Oh), K48-linked ubiquitin (kindly provided by Dr. Eugene Oh), FLAG (Thermo Fisher Scientific, MA1-91878), PSMD14 (Cell Signaling Technology, 4197), PSME3 (Proteintech, 14907-1-AP), H2B (Cell Signaling Technology, 12364), OTUD5 (Cell Signaling Technology, 20087), TRIP12 (Proteintech, 25303-1-AP), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (Proteintech, SA00001-2), and HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech, SA00001-1).

Techniques: Western Blot, Knockdown, Control, Irradiation, Immunofluorescence, Transfection, Fluorescence, Non-Homologous End Joining, Single Cell Gel Electrophoresis, Competitive Binding Assay, Labeling, Mutagenesis, Amplification, Two Tailed Test

Journal: bioRxiv

Article Title: Selective Elimination of TP53 Mutant Cells by Transcript-Activated Chromatin Shredding

doi: 10.64898/2026.05.08.723607

Figure Lengend Snippet: a, Prevalence of TP53 alterations in different cancers from TCGA Pan-Cancer Atlas studies. b, Schematic showing the protospacer flanking site (PFS) for Cas12a2 targeting. c, Growth curves of RPE1 WT and p53 mutant cells following Cas12a2 targeting of p53 R248Q mRNA. d , Growth competition assay between RPE1 WT and R248Q cells, both stably expressing Cas12a2. Images of merged red and green channels are shown. e, Fold-increase in cell number over 96 h following Cas12a2 targeting of the p53 R248Q mutant transcript in RPE1 p53 R175H control cells. f, Quantification of RNA levels using smFISH. Bar indicates the median. g, Immunoblots probing expression of DNA damage marker phospho-KAP1 48 h following Cas12a2 targeting of the p53 R248Q mutant transcript in RPE1 p53 R175H control cells. Statistics: ****p<0.0001; ns, non-significant, one-way ANOVA with Dunnett’s test.

Article Snippet: Immunoblotting was performed using the following antibodies diluted in TBS buffer supplemented with 0.1% Tween 20 and 5% milk powder or 3% BSA: p-KAP1 (1:1000, Bethyl Laboratories, A300-767A), p-H2A.X S139 (1:1000, Millipore, 05-636), GAPDH (1:1000, Santa Cruz, sc-365062), anti-Mouse HRP(1:5000, Invitrogen, 31430), anti-Rabbit HRP (1:5000, Invitrogen, 65-6120), anti-Rabbit IRDye800 (1:5000, Licor, 926-32211) and anti-Mouse IRDye680 (1:5000, Licor, 926-68070).

Techniques: Mutagenesis, Competitive Binding Assay, Stable Transfection, Expressing, Control, Western Blot, Marker

Journal: bioRxiv

Article Title: Selective Elimination of TP53 Mutant Cells by Transcript-Activated Chromatin Shredding

doi: 10.64898/2026.05.08.723607

Figure Lengend Snippet: a, Design of a gRNA (R280K g) targeting the p53 R280K mutant transcript. b, Design of a gRNA (E285K g) targeting the p53 E285K mutant transcript. c, Fold-increase in cell number over 96 h following Cas12a2 targeting of p53 R280K or E285K mutant transcripts. d, Immunoblots showing expression of DNA damage marker phospho-KAP1 in RPE1 p53 R280K, RPE1 p53 E285K and RPE1 WT cells 48 h following Cas12a2 targeting of the mutant transcripts. e, Design of gRNAs (M246I g1-6) targeting the p53 M246I mutant transcript. f, Trans -cleavage of purified genomic DNA by Cas12a2 with M246I gRNAs in the presence of the mutant target RNA or WT target RNA. g , Fold-increase in green intensity over 96 h following Cas12a2 targeting of the p53 M246I mutant transcript in NCI-H23 cells (p53 M246I) and U2OS cells (p53 WT) expressing Cas12a2-2A-EGFP. h, Analysis of mutation types in the TP53 coding sequence (CDS) from 16,708 tumor samples. Although Cas12a2 prefers an A-rich PFS , a consensus PFS sequence could not be built . However, we noticed that a single adenine nucleotide can sometimes serve as a PFS to activate Cas12a2 , and more adenine bases between the -2 to -4 position at the 3’ end of the protospacer can lead to higher Cas12a2 activity ( and ). 25.7% of TP53 mutations in patients are B-to-A substitutions (where B is G, C, or T) , which could serve as new or enhanced PFS for targeting, as demonstrated with the R248Q mutation . 17.1% of TP53 mutations are base insertions or deletions , offering opportunities for targeting similar to our approach with the EGFR in-frame deletion mutation . i, Frequency of the presence of A, AA or ANA within 24 nt at the 3’ end of TP53 mutations from 16, 708 tumor samples. Tumor sample data in c and d are from TCGA Pan-Cancer Atlas studies and MSK-CHORD studies. j, Cas12a2 targeting by transfecting mRNA encoding Cas12a2 and gRNAs into PC9 cells. Growth curves and fold-increase in cell number over 96 h are shown on the right. h, Characterization of LNPs used for the in vivo lung tumor treatment test. Statistics: **p<0.01; ****p<0.0001; ns, non-significant, one-way ANOVA with Dunnett’s test.

Article Snippet: Immunoblotting was performed using the following antibodies diluted in TBS buffer supplemented with 0.1% Tween 20 and 5% milk powder or 3% BSA: p-KAP1 (1:1000, Bethyl Laboratories, A300-767A), p-H2A.X S139 (1:1000, Millipore, 05-636), GAPDH (1:1000, Santa Cruz, sc-365062), anti-Mouse HRP(1:5000, Invitrogen, 31430), anti-Rabbit HRP (1:5000, Invitrogen, 65-6120), anti-Rabbit IRDye800 (1:5000, Licor, 926-32211) and anti-Mouse IRDye680 (1:5000, Licor, 926-68070).

Techniques: Mutagenesis, Western Blot, Expressing, Marker, Purification, Sequencing, Activity Assay, In Vivo