Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Colony formation by K562 cells expressing shRNA non-targeting (shNEG) or targeting mRNA of TIAL1 (shTIAR), TIA1 (shTIA-1), or FMR1 (shFMRP) that were collected from ex vivo hypoxic (1.5% O2) co-culture with HS-5 bone marrow stromal fibroblasts and treated with 1 μM imatinib (IM) or 50nM Talazoparib (BMN) added in two doses following the experimental scheme (left panel). Number of colonies in each of the 3 technical replicates from 3-4 independent biological experiments presented as % change relative to the untreated cells (dashed black line) set as 100 %. Student’s two-tailed t-test was used to compare two samples marked by the black line; #### or **** - p<0.0001, ns - p > 0.05. (B) Scheme explaining the experimental setup based on the subcutaneous implantation in mice of 3D printed scaffolds (photo taken with a Samsung mobile phone camera) seeded with human cells differentiated into osteoblasts and K562/luc cells with shNEG, shTIAR or shTIA-1, followed by IM treatment for 14 days. (C) Bioluminescence signal monitored in mice after 2 weeks of IM or vehicle treatment (timeline explained in (B) ) following luciferin injection, collected in the Burker’s Xtreme In-Vivo chamber for 30 sec, and overlaid on the mouse X-ray image. Scale presents the signal intensity of the color coding. (D) Sum of the signal intensity (P) collected per second (s) and area (mm 2 ) for each mouse analyzed (single dot) is presented. (E) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG or shTIAR, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days. (F) Weight of each xenograft isolated from mice (single dot) formed by K562/luc cells with shNEG or shTIAR (as in (E) ) presented as fold change of the weight mean value of xenografts from mice treated with vehicle; mean value indicated with the black line. The nonparametric two-tailed Mann-Whitney test was used for comparisons indicated by black lines underneath the exact significance ( P ) values are presented in the plot. (A,D,F) Two-way Anova was used to compare shTIAR upon IM versus other variants; @ - P < 0.002.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, shRNA, Ex Vivo, Co-Culture Assay, Two Tailed Test, Injection, In Vivo, Luciferase, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Scheme explaining experimental setup to determine proteomic changes using the quantitative BONCAT (QuaNCAT). K562 cells expressing shNEG or shTIAR growing in co-culture with HS-5 cells under hypoxia (1.5% O2) for 1.5 days were treated with imatinib (IM) for 18h before the bioorthogonal noncanonical amino acid tagging (BONCAT) of nascent proteins synthesized in cells for 4h. (B) Upper panel - number of genes and nascent proteins showing increased (UP; log₂ fold change [Log₂FC] ≥ 0.6) or decreased (DOWN; Log₂FC ≤ −0.6) abundance in IM-treated shTIAR cells compared with shNEG cells, with a significance threshold of p ≤ 0.05. In total, 2186 nascent proteins were quantified in the BONCAT experiment. Lower panel - scatter plot showing intensity-based absolute quantification (iBAQ), used as an estimate of relative protein molar abundance, plotted against Log₂FC values for BONCAT-identified proteins in shTIAR versus shNEG cells. Purple dots indicate hits with significant changes at both the protein and mRNA levels. Dashed vertical lines mark the Log₂FC thresholds of −0.6 and 0.6. (C) Functional annotation Gene Ontology Molecular Function enrichment analysis of UP (brown) or DOWN (purple) proteins with ClueGO/CytoScape, displaying proteins annotated with the term; only one side enriched terms with FDR < 0.05. (D) Lower panel - number of proteins that upon shTIAR are UP and DOWN regulated, for which mRNA was detected in the RNA immunoprecipitated (IP) in TIAR protein complexes (RIP), enriched in samples obtained with anti-TIAR antibody versus the same isotype non-binding antibody (ISO) (n=3647). Upper panel - example Image of Western blotting analysis of IP, with a sample of cell lysate used for IP loaded for reference (input). (E) Changes in the mRNA level, determined by real-time PCR and quantified using the ddCT method, expressed as log2 of change between IM-treated versus untreated cells, detected in samples from whole cells (upper panel) and anti-TIAR RIP (lower panel). Mean of 3 independent experiments with ± range is presented. Student’s t-test two-way was used to compare the difference between IM to C; * p ≤ 0.05, ** p≤0.005. (F) Number of genes identified in anti-TIAR RIP that show significant changes in intron retention (RI) or cassette exon alternative splicing (CE) in shTIAR versus shNEG alternative splicing analysis of RNA from IM-treated cells. Number of genes in the intersection provided above the bar; comparisons indicated by the black dot. (G) Sashimi plot (middle panel) demonstrating splicing of EIF4A2 mRNA within the region encompassing exons 8-11 and 3’UTR in RNA from anti-TIAR RIP. Alternative splice site usage marked by the purple line, and the percent usage ± ME (n=3) in numbers by the lines, reads coverage from 0-145 shown in grey, alternatively spliced exon marked by shaded yellow. Gene region scheme in the top. (H) Percent transcripts with TIAR-dependent alternative exon inclusion in K562 cells from xenografts treated with IM. Student’s t-test was used to compare results from three experiments (each dot) for the mean value marked with a thick black line; * p = 0.021.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, Co-Culture Assay, Synthesized, Quantitative Proteomics, Functional Assay, Immunoprecipitation, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Alternative Splicing

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Expression level of genes analyzed at the scdbm for CD34 + cells subtype; patient classification explained in . (B-H) Impact of shTIAR compared to shNEG analyzed in K562 cells that were co-cultured with HS-5 cells under hypoxia (1.5%O2) 2 days before initiation of 18h culture with imatinib (IM) treatment or without (C). (B) Protein level in whole cell extracts analyzed by Western blot, representative images of immunoblots (n=3) presented. (C) Mitochondrial (mit.) membrane potential measured using JC-1 probe; values for signal from probe aggregates (red) in polarized mitochondria expressed as % of signal from the probe in the cells (n=3), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) used as a control. (C,D,F,H) Bars represent the mean of values from independent experiments (indicated by dots) with ± SD. Student’s t-test was used to determine the significance of the difference shTIAR vs shNEG (#) and IM vs C (*); * - p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.001 (D) Lipid peroxidation measured with click-it chemistry by flow cytometry. Fluorescence intensity GeoMean expressed as fold change of value in the untreated shNEG cells (n=5). (E-H) Presentation on the cluster of cell differentiation (CD) surface protein markers CD45 and CD235a on K562 cells with shNEG or shTIAR isolated from co-culture established in hypoxia (1.5%O2) a day before initiation of treatment with IM for 48h (E-G) or FACS-sorted K562/luc GFP positive cells from xenografts (H) . (E-F,H) Percentage of parental live cell subpopulations that are: double positive for CD45 and CD235a (CD235a&CD45), positive only for CD45, or only for CD235a, or negative for both CD markers. Representative scatter plots in (E) , summary of independent co-culture experiments (n=4) in (F) . (G) Fluorescence intensity of surface CD36 protein staining in the fraction of cells positive for CD235a or CD45. Numbers correspond to fold change in shTIAR to shNEG ± ME (n=2). (H) Percent GFP and hCD45-positive cells from different xenografts (n=4) that are positive for CD235a. Two-way ANOVA test was used to for comparison of shNEG_C to other variants (@), and p=0.0046.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, Cell Culture, Western Blot, Membrane, Control, Flow Cytometry, Fluorescence, Cell Differentiation, Isolation, Co-Culture Assay, Staining, Comparison

Journal: PeerJ

Article Title: Integrated analysis of programmed cell death-related genes identifies CORO1A as an apoptosis-associated gene in acute myeloid leukemia

doi: 10.7717/peerj.21303

Figure Lengend Snippet: (A) CORO1A expression levels in HS-5, HL-60 and THP-1 cells. (B) CORO1A expression levels in HL-60 and CORO1A knockdown HL-60 cell lines. (C) CORO1A expression levels in THP-1 and CORO1A knockdown THP-1 cell lines. (D) CCK-8 analysis of CORO1A knockdown HL-60 and THP-1cell lines. (E) Bcl-2 and Bax expression levels in CORO1A knockdown HL-60 cell line. (F) Bcl-2 and Bax expression levels in CORO1A knockdown THP-1 cell line. (G) Annexin V staining analysis of CORO1A knockdown HL-60 cell line. (H) Annexin V staining analysis of CORO1A knockdown THP-1cell line.

Article Snippet: Human HS-5, HL-60, and THP-1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Knockdown, CCK-8 Assay, Staining

Journal: bioRxiv

Article Title: Efficacy evaluation of glasedgib Sonic Hedgehog pathway inhibition with or without inotuzumab in B-ALL cells using a new co-culturing system model and a validated chemosensitivity assay

doi: 10.64898/2026.05.07.723573

Figure Lengend Snippet: Primary bone marrow cells co-cultured with HS-5 marrow stromal cells. (A) Visualization of the culture in 1 μM Hoechst 33258 (blue) and 0.5 μM NucView (green, indicating activated caspase 3 in cells undergoing apoptosis. (B) Image analysis inverting the blue channel from part A to differentiate HS-5 cells (large diffuse; red arrow) from bone marrow cells (small, roundish, and intense).

Article Snippet: Patients’ cells were cultured in vitro with support of a feeder cell layer HS-5 stromal cells (ATCC CRL-11882).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Efficacy evaluation of glasedgib Sonic Hedgehog pathway inhibition with or without inotuzumab in B-ALL cells using a new co-culturing system model and a validated chemosensitivity assay

doi: 10.64898/2026.05.07.723573

Figure Lengend Snippet: (A) Experimental design: primary B-ALL blasts were recovered from cryostorage and briefly allowed to recover in co-culture with HS-5 stromal support. Viable blasts were plated for chemosensitivity testing in co-culture and exposed to treatments for four days. Hematopoietic cells were mechanically isolated and assessed for viability using CellTiter-Glo. (B) Relative viability: dose-dependent responses to glasdegib are in seen within the hematopoietic compartment of co-cultured cells (Patients 1 and 2) but not in HS-5 stromal cells alone. Unique patients have differential responses to inotuzumab alone, but co-treatment sensitizes blasts to the effect of glasdegib.

Article Snippet: Patients’ cells were cultured in vitro with support of a feeder cell layer HS-5 stromal cells (ATCC CRL-11882).

Techniques: Co-Culture Assay, Isolation, Cell Culture

Journal: bioRxiv

Article Title: Efficacy evaluation of glasedgib Sonic Hedgehog pathway inhibition with or without inotuzumab in B-ALL cells using a new co-culturing system model and a validated chemosensitivity assay

doi: 10.64898/2026.05.07.723573

Figure Lengend Snippet: The SHH gene expressions by four primary B-ALL patient samples in different culture conditions. Each panel represents a different patient sample, and each color corresponds to the SHH gene indicated in the legend. The x-axes represent the treatment conditions: negative control (Co DMSO), 20 μM glasdegib (Co Glas 20), and 20 μM glasdegib + 10 ng/mL inotuzumab (Co Glas 20 INO). The y-axes represent the expression of GLI1, GLI3, SMO , and PTCH1 after four days of HS-5 stromal cell co-culture. All expression values were represented by method with GAPDH as the internal control and treatment with DMSO as the fold-change calibrator.

Article Snippet: Patients’ cells were cultured in vitro with support of a feeder cell layer HS-5 stromal cells (ATCC CRL-11882).

Techniques: Negative Control, Expressing, Co-Culture Assay, Control

Journal: Cancers

Article Title: Extracellular Enolase-1 Promotes CAF-Associated Stromal Reprogramming via the Plasmin/TGF-β Axis in Multiple Myeloma

doi: 10.3390/cancers18091467

Figure Lengend Snippet: HuL001 reduces CAF-like differentiation, glycolytic activity, and secretion of IL-6 and VEGF in MM-educated BMSCs. ( A – C ) NOD.Cg-Prkdc scid Il2rg tm1Vst /Vst mice bearing KMS-11/BTZ xenografts were treated with vehicle or HuL001 (30 mg/kg, i.p., twice weekly) starting at ~100 mm 3 . FAP and HK2 were assessed in five representative tumors per group, with GAPDH as a loading control. Panels ( B , C ) show the quantified FAP and HK2 levels corresponding to panel ( A ). ( D ) Experimental design: HS-5 BMSCs (adherent culture) were directly co-cultured with KMS-11/BTZ cells (suspension culture) at a 1:2 ratio with or without either HuL001 (10 µg/mL) or human IgG1 (10 µg/mL) for 5 days. Subsequently, suspended KMS-11/BTZ cells were removed, and adherent MM-educated BMSCs (referred as CAFs) were collected for immunoblot analysis or secretion assays. ( E , F ) Representative immunoblots from one of three independent biological experiments. Panel ( E ) shows FAP and HK2, and panel ( F ) shows FSP1, PDGFRβ, and α-SMA. ( G , K ) Densitometric quantification of FAP and HK2 immunoblot signals corresponding to the experiments represented by panel ( E ), based on three independent biological experiments. ( H – J ) Densitometric quantification of FSP1, PDGFRβ, and α-SMA immunoblot signals corresponding to the experiments represented by panel ( F ), based on three independent biological experiments. Quantified protein levels were normalized to GAPDH, and relative expression levels were calculated by comparison with un-educated BMSCs, which were set to 1.0. ( L – N ) After removal of suspended KMS-11/BTZ cells, adherent BMSCs were incubated with fresh culture medium for 2 h, and the secretion of ( L ) lactate, ( M ) IL-6, and ( N ) VEGF was measured. The results were normalized to the cell number in each group. Data in panels ( G – N ) are presented as the mean ± standard deviation from at least three independent biological experiments. Statistical analyses were performed using two-sided unpaired Student’s t -test or one-way analysis of variance with Tukey’s post hoc test, as appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Original Western blot images corresponding to the blots shown in this figure are provided in .

Article Snippet: The immortalized human BMSCs HS-5 (cat. no. CRL-3611) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were authenticated using STR profile analysis at ATCC.

Techniques: Activity Assay, Control, Cell Culture, Suspension, Western Blot, Expressing, Comparison, Incubation, Standard Deviation

Journal: Cancers

Article Title: Extracellular Enolase-1 Promotes CAF-Associated Stromal Reprogramming via the Plasmin/TGF-β Axis in Multiple Myeloma

doi: 10.3390/cancers18091467

Figure Lengend Snippet: HIF-1α contributes to glycolytic remodeling and modulates FAP expression in MM-educated BMSCs. HS-5 BMSCs were transfected with HIF-1α siRNA or control siRNA (scramble) for 3 days, and then co-cultured with KMS-11/bortezomib cells at a BMSC:MM ratio of 1:2. After removing the suspended MM cells, the adherent BMSCs were analyzed for ( A ) HIF-1α and HK2 at day 1 and ( B ) FAP at day 5, or ( F ) were incubated in fresh medium for 2 h to assess lactate secretion. Panels ( A , B ) show representative immunoblots from one of three independent biological experiments, with GAPDH used as a loading control. Panels ( C , D ) show densitometric quantification from the same experimental set represented in panel ( A ), based on three independent biological experiments. Panel ( E ) shows densitometric quantification from the same experimental set represented in panel ( B ), based on three independent biological experiments. The signals were normalized to GAPDH, and relative expression levels were calculated by comparison with un-educated BMSCs (MM−, scramble), which were set to 1.0. The results in panels ( C – F ) are presented as the mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance with Tukey’s post hoc test. * p < 0.05, *** p < 0.001. Original Western blot images corresponding to the blots shown in this figure are provided in .

Article Snippet: The immortalized human BMSCs HS-5 (cat. no. CRL-3611) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were authenticated using STR profile analysis at ATCC.

Techniques: Expressing, Transfection, Control, Cell Culture, Incubation, Western Blot, Comparison, Standard Deviation

Journal: Cancers

Article Title: Extracellular Enolase-1 Promotes CAF-Associated Stromal Reprogramming via the Plasmin/TGF-β Axis in Multiple Myeloma

doi: 10.3390/cancers18091467

Figure Lengend Snippet: Extracellular ENO1 promotes cancer-associated fibroblast-like differentiation of BMSCs through the plasmin/TGF-β axis. ( A , G , J ) HS-5 BMSCs were cultured in 50% CM derived from KMS-11/bortezomib cells (MM CM) with or without ENO1 protein, TXA (10 mM) and the TGF-β receptor kinase inhibitor SB431542 (10 nM) for 1 day. ( B ) HS-5 BMSCs were transfected with hexokinase 2 small interfering RNA or scramble control, cultured in 50% MM CM for 3 days, and then treated with ENO1 protein for 1 additional day before immunoblot analysis. In panels ( A , B , G , J ), the left panels show representative immunoblots for FAP, whereas the right panels show densitometric quantification of FAP immunoblot signals from the same experimental sets. FAP signals were normalized to GAPDH, and relative expression levels were calculated by comparison with untreated cells, which were set to 1.0. Quantification in panels ( A , B , G ) was based on two independent biological experiments, whereas quantification in panel ( J ) was based on three independent biological experiments. Data in the right panels of ( A , B , G , J ) are presented as the mean ± standard deviation. ( C – F , I ) HS-5 BMSCs were cultured in 50% MM CM with or without ENO1 protein, HuL001 (100 µg/mL), hIgG1 (100 µg/mL), and TXA (10 mM) for 2 days. Supernatants were collected to measure ( C ) lactate, ( D ) interleukin-6, ( E ) vascular endothelial growth factor and ( F , I ) active transforming growth factor-β1. The results were normalized to the cell number in each group. ( H ) HS-5 BMSCs were cultured in 50% MM CM with or without ENO1 protein, HuL001 (100 µg/mL), and hIgG1 (100 µg/mL), followed by measurement of plasminogen receptor activity. Data in panels ( C – F , H , I ) are presented as the mean ± standard deviation from three independent biological experiments. Statistical analyses were performed using one-way analysis of variance with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Original Western blot images corresponding to the blots shown in this figure are provided in .

Article Snippet: The immortalized human BMSCs HS-5 (cat. no. CRL-3611) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were authenticated using STR profile analysis at ATCC.

Techniques: Cell Culture, Derivative Assay, Transfection, Small Interfering RNA, Control, Western Blot, Expressing, Comparison, Standard Deviation, Activity Assay