Journal: bioRxiv

Article Title: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

doi: 10.1101/2023.01.01.522017

Figure Lengend Snippet: (A) Expression of surface ERBB4 by FACS in parental (grey) and resistant (red) lines. Dashed black line for negative control (neg-cnt). Data derived from two independent experiments. (B) Secretion of HBEGF in parental (grey) and resistant (red) by ELISA. Data derived from three independent experiments. (C) Expression levels of surface CD19 (x-axis, PE-conjugated) and intracellular HBEGF (y-axis, APC-conjugated) by FACS in parental (left) and resistant (right) cells, percentage correspond to percentages of total viable cells. Data representative of two independent experiments. (D) Levels of protein phosphorylation by immunoblot in parental (grey) and resistant (red) cells. Values correspond to average of protein quantification normalized to GAPDH levels in three independent experiments. Error bars represent standard error of the mean. (E) Quantification of AKT phosphorylation by immunofluorescence, error bars represent standard error of the mean. Data derived from at least three independent experiments. P values from t-test, statistically significant for p< 0.05. (E) Detail of p-AKT expression by immunofluorescence in parental (PAR, top) and resistant (RES, bottom) upon DMSO (control, left) or 1μM idelalisib (right). (G) Cell viability by MTT assay of parental cells (PAR, black line) exposed to idelalisib in presence or not of conditioned medium from Karpas1718 idelalisib-resistant (RES, red line), cultured for 48h, PAR + RES-cond RPMI, dashed black line) or stimulated with 30ng/ml of recombinant HBEGF (dashed red line). Data derived from the average of three independent experiments. P values from a moderated t-test, statistically significant for p< 0.05.

Article Snippet: ELISA (Human HB-EGF Quantikine ELISA Kit, R&D Systems) was performed according to the manufacturer’s protocols.

Techniques: Expressing, Negative Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Immunofluorescence, Control, MTT Assay, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

doi: 10.1101/2023.01.01.522017

Figure Lengend Snippet: (A) Small interfering RNAs were used for gene expression silencing of ERBB2 alone (yellow dashed line), ERBB4 alone (red dashed line) or concomitant silencing of ERBB2 and ERBB4 (brown dashed line). Black lines for resistant control. (B) miRNAs mimics were used for miR-29c expression (yellow dashed line), let-7c (red dashed line) or miR-30c (brown dashed line). Black lines for resistant control. Drug sensitivity was evaluated by MTT assay in resistant Karpas1718 cells for the PI3K inhibitors idelalisib, umbralisib, copanlisib and duvelisib. Data correspond of two independent experiments, error bars represent standard deviation of the mean. P values from a moderated t-test, statistically significant for p< 0.05. (C) Levels of HBEGF secretion by ELISA in resistant cells upon miRNA mimics for miR-29c (yellow), let-7c (red) or control (grey). Data derived from three independent experiments. P values from a moderated t-test, statistically significant for p< 0.05.

Article Snippet: ELISA (Human HB-EGF Quantikine ELISA Kit, R&D Systems) was performed according to the manufacturer’s protocols.

Techniques: Gene Expression, Control, Expressing, MTT Assay, Standard Deviation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: bioRxiv

Article Title: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

doi: 10.1101/2023.01.01.522017

Figure Lengend Snippet: (A) HBEGF secretion was evaluated in the serum of CLL samples at the end of the treatment by ELISA (Luminex, R&D Systems). (A) Patients responding (responders, blue) or non-responding (non-responders, red) to idelalisib were compared by t-test. (B) HBEGF secretion in the serum of patients treated with ibrutinib was compared at the time of progression (red) with baseline levels previously of ibrutinib treatment (pre-treatment, blue). P for adjusted p-value from a t-test. MFI: mean fluorescence intensity.

Article Snippet: ELISA (Human HB-EGF Quantikine ELISA Kit, R&D Systems) was performed according to the manufacturer’s protocols.

Techniques: Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence