pladienolide b  (Tocris)

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a) Cell viability assay in cells treated or not for 72 hours with increasing concentrations of pladienolide B (nM). Mean ± SD. n = 3. (b) Quantification of apoptosis (%) by flow cytometry in cells treated with 5 nM pladienolide B for indicated times. Mean ± SD. n = 3. (c) Clonogenic assay in H460S/R cells treated or not for 14 days with 1 nM pladienolide B or 1 µM cisplatin. Lower panel: number of colonies with number in untreated condition being arbitrarily assigned to 100% survival. Mean ± SD. n = 3. (d) Spheroids from H460S/R cells treated with complete medium and DMSO as control, or 0.5 nM or 1 nM pladienolide B for the indicated days. Upper panels: representative images of spheroids at day 14. Lower panel: spheroids area (µm 2 ). Mean ± SD. n = 5 spheroids/condition. A representative experiment of 3 independent ones. (e) Representative immunoblots of SF3B1 in H460S/R or A549S/R cells. GAPDH was used as a loading control. n = 3. (f) H460R or A549R cells were transfected for 72 hours with a control siRNA or two distinct siRNA targeting Sf3b1 mRNA as indicated. Left panels: representative immunoblots of SF3B1. GAPDH was used as a loading control. Right panels: quantification of apoptosis (%). Mean ± SD. n = 3. (a, b, c, d, f) Unpaired t test. * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001, ns: not significant. (g) Overall Rate Response (ORR) at Day 20 for all NSCLC PDXs treated or not (control) with 2.5 mg/kg or 5 mg/kg pladienolide B (ip, Day 1-Day 4-Day 8-Day 11). Two-tails Mann-Whitney t test. ** p< 0.01. (h) Probability of progression (Relative Tumor Volume = 2) in control and pladienolide B-treated PDXs. n = 7 (LCIM1 is not included). Log-rank t test. ** p < 0.01.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: Viability Assay, Flow Cytometry, Clonogenic Assay, Control, Western Blot, Transfection, MANN-WHITNEY

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet:

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: Inhibition, Mutagenesis

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a) H460S/R or A549S/R cells were treated or not with 5 nM pladienolide B for indicated times. Cell cycle distribution (%) in the different phases of the cell cycle. Mean ± SD. n = 3. Unpaired t test. * p< 0.05, ** p< 0.01, **** p< 0.0001. (b) Percentage of EdU-positive H460R cells in untreated or pladienolide B condition. Mean ± SD. n = 3. Unpaired t test. * p< 0.05. (c, d, e) H460R and H460S cells were treated or not with 5 nM pladienolide B for 6 hours. Upper panels: representative immunofluorescence stainings of RPA32 (c), γH2AX (d), or phospho-53BP1(Ser1778) (e). DAPI was used to counterstain the nucleus. Scale bar = 10 µm. Lower panels: quantification of the number of RPA32 (c), γH2AX (d), or phospho-53BP1(Ser1778) (e) foci/nucleus. Mean ± SD. n = 3. Unpaired t test. ** p< 0.01, *** p< 0.001, **** p<0.0001. (f) Upper panels: representative immunoblots of γH2AX protein in H460S/R cells treated with 5 nM pladienolide B for indicated times. KU80 was used as a loading control. Lower panels: densitometric quantification (fold change) of γH2AX signal normalized to KU80 signal. Mean ± SD. n=3. Mann-Whitney t test. * p< 0.05. (g, h) H460R cells were treated or not for 6 hours with 5 nM pladienolide B. (g) SIRF analysis of PCNA recruitment at nascent replication forks. Lefts panels: representative images. Right panel: quantification of EdU-PCNA fluorescent intensity (ratio signal intensity/area)/nucleus. Mean. n = 3. Unpaired t test. **** p<0.0001. (h) PCNA immunoblot. Tubulin was used as a loading control.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: Immunofluorescence, Western Blot, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a) Immunoblots of the indicated proteins in H460S/R or A549S/R cells treated or not with 5 nM pladienolide B for 6 hours. KU80 was used as a loading control. (b) Densitometric quantification (fold change) of P-DNA-PKcs(Ser2056), P-RPA32(Ser4/8) or P-ATR(Thr1989) signal normalized to KU80 signal. Mean ± SD. n=3. (c) H460R cells were co-treated (+) or not (-) with 5 nM pladienolide B for 4 hours in the presence or absence of 25 µM DRB. Representative immunoblots of the indicated proteins. GAPDH was used as a loading control. n = 3. (d) H460R cells were co-treated (+) or not (-) with 5 nM pladienolide B for 6 hours in the presence or absence of 25 µM DRB. Left panels: representative immunofluorescence stainings of γH2AX in each condition. DAPI was used to counterstain the nucleus. Scale bar = 10 µm. Right panel: quantification of the number of γH2AX foci/nucleus. Mean. n = 3. (e, f) H460S/R or A549S/R cells were treated or not with 5 nM pladienolide B for the indicated times. (e) Upper panels: Immunoblots of the indicated proteins. KU80 was used as a loading control. Lower panels: densitometric quantification (fold change) of DNA-PKcs or ATR signal normalized to KU80 signal. Mean ± SD. n=3. (f) RT-qPCR (fold change) of PRKDC or ATR mRNA level. Mean ± SD. n = 3. All, unpaired t test. * p< 0.05, ** p< 0.01, *** p< 0.001, **** p<0.0001, ns: not significant.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: Western Blot, Control, Immunofluorescence, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a) Left panel: total number of differentially Expressed Genes (DEGs) based on RNA-Seq studies in H460S/R cells treated with 3 nM pladienolide B for 8 hours as compared to untreated condition. Genes with p value ≤ 0.05 and absolute Log2 FC ≥ 0.4 were considered as significant. Right panel: number of DEGs either down- or up-regulated. (b) Number of DEGs either down- or up-regulated in H460S/R cells according to absolute Log2 FC ≥ 1 and p value ≤ 0.05. (c) DEGs belonging to the DNA repair pathways full network (WikiPathways, WP4946) in H460S/R cells. (d, e) Number of total splicing events, according to each type of alternative splicing, differentially regulated in H460 parental or resistant cells treated with 3 nM pladienolide B for 8 hours as compared to untreated condition. (d) Number of differential splicing events with p value ≤ 0.05 and absolute deltaPSI ≥ 10%. (e) Number of differential splicing events with p value ≤ 0.05 and absolute deltaPSI ≥ 20%. (f) Upper panel: number of specific and common Differentially Spliced Genes (DSGs) with absolute deltaPSI ≥ 20% in H460R and H460S cells. Lower panel: Gene Ontology Analysis on common DSGs (n = 2458). (g) RT-PCR validation of exon skipping events impacting genes involved in DNA repair pathways in H460S/R cells treated or not with 5 nM pladienolide B for indicated times. Representative agarose gels of amplified products. Numbers indicate amplicon size (in base pairs). n = 3.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: RNA Sequencing, Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Amplification

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a) Quantification of DSB-induced homologous recombination in H1299 clones stably expressing the pDR-GFP (pBL174) plasmid. When these cells are transfected with the pBL133 plasmid encoding the I-SceI restriction enzyme, efficient recombination restores a functional GFP coding sequence. (b) H1299-pBL174 cells were transfected with pcDNA3.1 or pBL133 for 24 hours and treated (pBL133+PDB) or not (pBL133) with 5 nM pladienolide B during 48 additional hours. Left panels: representative FL1-H versus FSC-H dot plots of GFP-positive (FL1-H +) and –negative (FL1-H -) cells. Right panel: GFP-positive cells (%). Mean ± SD. n = 3. Unpaired t test. ** p<0.01, ns: not significant. (c) A549 LUAD cells stably expressing the pBL230 plasmid were generated. This plasmid contains genes encoding the membrane antigens CD4 and CD8. CD8 is not expressed as it is in inverted orientation, and CD4 is not expressed because it is too far from the promoter. Two cleavage sites for I-SceI are present in non-coding sequences, which are in direct orientation generating cohesive ends between the two sites. When two DSBs are produced by I-SceI, rejoining of the DNA ends by exclusion or inversion leads to the expression of CD4 or CD8, respectively. (d) Quantification of DNA repair by c-NHEJ in A549-pBL230 clones transfected with either pcDNA3.1 or pBL133 plasmid and treated or not with 5 nM pladienolide B for 24 or 48 hours. Left panel: western blotting of I-SceI-HA in transfected cells treated or not with pladienolide B for 48 hours. Right panel: CD4-positive cells (%). Mean ± SD. n = 3. Mann-Whitney t test. * p< 0.05. (e) G2PCC in H460R cells treated or not with 5 nM pladienolide B for indicated times. Left panels: representative images of G2PCC showing chromosome breaks and exchanges. Right panels: quantification of the number of breaks or exchanges per 100 G2PCC. Mann-Whitney t test. * p< 0.05, ** p<0.01, ns: not significant. (f) Clonogenic survival assay. H460S or H460R cells were treated or not with 1 nM pladienolide B for 10 days in the presence or absence of 5 µM BO2 a RAD51 inhibitor, which was the only drug removed after 24 hours treatment, 10 µM 6-OH-DOPA, a RAD52 inhibitor, or 10 µM olaparib, a PARP1/2 inhibitor. The number of colonies obtained in non treated condition was arbitrarily assigned to 100% survival. Mann-Whitney t test. * p< 0.05, ** p<0.01, *** p< 0.001, **** p<0.0001.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: Homologous Recombination, Clone Assay, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Functional Assay, Sequencing, Generated, Membrane, Produced, Western Blot, MANN-WHITNEY, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a, b) H460S/R or A549S/R cells were treated with 5 nM pladienolide B for indicated times. RT-PCR analysis of skipping of MLH3 -Ex8 (a) or MLH3 -Ex5 (b). Left panels: representative agarose gels of amplified products. Numbers indicate amplicon size (in base pairs). Right panels: densitometric quantification of exon 8 or 5 exclusion (%). Mean ± SD. n = 4 (H460S/R) or n = 3 (A549S/R). (c, d) H460R or A549R cells were transfected with control siRNA or two distinct siRNAs targeting SF3B1 for 72 hours. (c) Left panels: RT-qPCR (fold change) of SF3B1 mRNA level. Mean ± SD. n = 3. Right panels: RT-PCR analysis of MLH3 -Ex8 or MLH3 -Ex5 skipping. Upper panels: representative agarose gels of amplified products. Numbers indicate amplicon size (in base pairs). Lower panels: densitometric quantification of exon 8 exclusion (%). Mean ± SD. n = 4. (d) Upper panel: representative immunoblots of SF3B1 or MLH3 protein levels. GAPDH was used as a loading control. Lower panels: densitometric quantification of SF3B1 or MLH3 protein level according to GAPDH. Number obtained in non treated condition was arbitrarily assigned the value 1. Mean ± SD. n = 3. All, Mann-Whitney t test. **p< 0.01, *** p<0.001, **** p<0.0001. (e) Correlation between SF3B1 mRNA level and usage of MLH3 -Ex8 or MLH3 -Ex5 in lung adenocarcinoma patients.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Control, Quantitative RT-PCR, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a-g) H460R or A549R cells were treated (+) or not (-) for 24 hours with 5 nM pladienolide B in the presence or absence of 10 µM (a) or 50 µM (a, b, c, d, e, f, g) cisplatin as indicated. (a) Quantification of apoptosis (%) by flow cytometry. Mean ± SD. n = 3. (b) Representative immunoblots of indicated proteins. α-tubulin was used as a loading control. n = 3. (c) Densitometric quantification of the signal obtained for the indicated proteins according to the signal obtained with α-tubulin. Number obtained in non treated condition was arbitrarily assigned the value 1. Mean ± SD. n = 3. (d) RT-PCR analysis of skipping of MLH3 -Ex8 or MLH3 -Ex5. Representative agarose gels of amplified products. Right numbers indicate amplicon size (in base pairs). (e) Densitometric quantification of exon 8 exclusion (%). Mean ± SD. n = 3. (f) RT-qPCR (fold change) of MLH3 mRNA level. (g) Upper panels: representative immunoblots of MLH3 protein levels. α-tubulin was used as a loading control. Lower panels: normalized densitometric quantification (fold change) of MLH3 signal according to α-tubulin signal. Number obtained in untreated condition was arbitrarily assigned the value 1. All, Mann-Whitney t test. * p< 0.05, ** p< 0.01, *** p< 0.001, ns: not significant.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: Flow Cytometry, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, MANN-WHITNEY

Journal: bioRxiv

Article Title: The SF3B1 inhibitor pladienolide B massively inhibits DNA damage signaling and repair and counteracts resistance to platinum salts in Non-Small Cell Lung Cancer

doi: 10.64898/2026.02.17.706284

Figure Lengend Snippet: (a) Overall Rate Response (ORR) of all NSCLC PDXs (n = 25) treated with 4 mg/kg cisplatin, 5 mg/kg pladienolide B or a combination of both as compared to vehicle condition. Two-tails Mann-Whitney t test. ** p< 0.01, *** p< 0.001. (b) Probability of tumor progression (Relative Tumor Volume = 2) in control, pladienolide B-, cisplatin- or pladienolide B + cisplatin-treated PDXs. n = 7. Log-rank test. * p< 0.05, ** p < 0.01, *** p< 0.001. (c) LCF26 and ML1LC2 PDXs were treated with vehicle (ctrl), 4 mg/kg cisplatin, 5 mg/kg pladienolide B, or a combination of both. Left panels: immunoblots of DNA-PKCs and ATR. 3 PDXs/condition. GAPDH was used as a loading control. Right panels: normalized expression of DNA-PKcs or ATR according to GAPDH. Mann-Whitney t test. * p< 0.05, ** p< 0.01. (d) RT-PCR analysis of skipping of MLH3 -Ex8 in each PDX (3 distinct mice/PDX). Representative agarose gels of amplified products. Right numbers indicate amplicon size (in base pairs). Below numbers indicated the percentage of exon 8 exclusion. Mean ± SD.

Article Snippet: Pladienolide B (cat#sc-391691, Santa Cruz) and 5,6-dichlorobenzimidazole 1-φ3-D-ribofuranoside (DRB) (cat#D1916, Sigma Aldrich) were prepared at 10 mM in stock solution, stored at -80°C and dilute just before use in medium.

Techniques: MANN-WHITNEY, Control, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: bioRxiv

Article Title: The splicing paralogues SNRPB and SNRPN control differential metabolic states

doi: 10.64898/2026.02.11.705284

Figure Lengend Snippet: (a, b, c, d, e, f, h, i, j) Cropped immunoblots of SNRPB - and SNRPN -cells under various conditions. RNA Pol2, PCNA, GAPDH and Coomassie serve as loading controls. HA-antibody shows overall protein stability differences. Monoclonal SmB/B’/N antibody (clone 12F5) shows detection differences for SNRPN under certain conditions. All experiments were performed at least twice with similar results. For control conditions, the solvent (DMSO, Ethanol, Water) in which the inhibitors are dissolved were added to the media. (a) Truncation mutant of SNRPB - amino acids 1-190 versus 1-231 (full-length) (b) SNRPB/N mutants encoding only amino acids 190 until C-terminal end. (c) SNRPB/N -cells treated 27 hours with 5 μM of PRMT4/CARM1 (left) or 6 days with 10 μM of PRMT5 inhibitor (right). (d) SNRPB/N -cells treated with translation inhibitors Cycloheximide (CHX, 4 hours, 100 μg/mL) or Puromycin (PURO, 20 μM) (e) SNRPB/N -cells treated with mTOR inhibitor INK128 (320 μM). The pAKT blot serves as a positive control for effective treatment. (f) SNRPB/N -cells treated with splicing inhibitor Pladienolide B (PlaB, 16 hours, 100 nM). Effective treatment was evaluated by RT-qPCR, see Extended Data Fig. 5c. (g) Quantification of post-translational modifications (PTMs) by mass spectrometry, as in , including CHX-treated conditions. The experiment was performed together, so the same datapoints from are also shown here (control condition). Each replicate IP sample was split and digested with either elastase (circles) or trypsin (triangles) to generate peptides for mass spectrometry. (h) SNRPB/N -cells treated with NMD inhibitor hSMGi-11e (24 hours, 0.6 μM). Effective treatment was evaluated by RT-qPCR, see Extended Data Fig. 5i. (i) SNRPB/N -cells grown in regular media (1x), media containing no arginine (left, 0x) or no Lysine (right, 0x) or 10-fold higher arginine (left, 10x) or Lysine (right, 10x) concentrations. Treatment was conducted for 16 hours. (j) SNRPB/N -cells grown in regular media (lane 1), media containing L-Glutamine instead of L-Alanyl-L-Glutamine (lane 2), regular media (lane 3) or regular media with reduced glucose content (lane 4). Treatment was conducted for 16 hours.

Article Snippet: A 1 mM stock of Pladienolide B (Santa Cruz Biotechnology, sc-391691) in DMSO was diluted to 100 nM for 16-hours.

Techniques: Western Blot, Control, Solvent, Mutagenesis, Positive Control, Quantitative RT-PCR, Mass Spectrometry