Journal: bioRxiv

Article Title: Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure

doi: 10.1101/2022.07.31.502226

Figure Lengend Snippet: A) Pedigrees of three families with homozygous DNAJB4 variants. Affected patients are in black. The genotype of the patient is represented within the pedigree. B) ClustalW alignment of DNAJB4 amino acid sequence from multiple species demonstrating conservation at R25 and L262. C) Schematic of the DNAJB4 denoting the J, G/F rich and C-terminal domains and the location of the identified variants. D) Lower extremity imaging of the probands from family A (MRI) and family B (CT scan).

Article Snippet: Antibodies used were the following: anti-rabbit GAPDH (Cell Signaling, 2118), anti-mouse Desmin (Dako, M0760), anti-rabbit DNAJB6 (Abcam, ab198995), anti-mouse HspA1 (Enzo, ADI-SPA-810), anti-rabbit α-actinin (Abcam, ab68167), anti-rabbit Myotilin (Abcam, ab78492), anti-rabbit Synemin (Bioss, bs-8555R), anti-rabbit αβ-crystallin (Enzo, ADI-SPA-223), anti-rabbit DNAJB4 (proteintech, 13064-1-AP) and anti-human DNAJB4 (Santa Cruz, sc-100711).

Techniques: Sequencing, Imaging, Computed Tomography

Journal: bioRxiv

Article Title: Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure

doi: 10.1101/2022.07.31.502226

Figure Lengend Snippet: A) Immunoblot of skeletal muscle tissue lysates from control patient muscle or patient A:I carrying homozygous p.Lys286Ter variants in DNAJB4 with an anti-DNAJB4 antibody. B) Immunoblot from lysates of control patient fibroblasts or primary fibroblasts from patient A:I (p.Lys286Ter) and B:I (p.Leu262Ser) with anti-DNAJB4 or anti-actin antibodies. C) Isogenic 293T cells stably expressing V5 tagged DNAJB4-WT, DNAJB4-R25Q, DNAJB4-F90L, DNAJB4-L262S or DNAJB4-K286Ter under a tetracycline inducible promoter were treated with tetracycline for 48 hours and then lysates were harvested for five days following tetracycline removal. Lysates were then immunoblotted for V5 (DNAJB4) and GAPDH. D) Two-photon confocal microscopy of mouse footpad muscle electroporated with GFP tagged DNAJB4-WT or DNAJB4-R25Q (green) with associated second harmonic generation imaging (SHG). DNAJB4 is at the Z-disc as SHG marks the A-band. E-F) Immunofluorescent images of a single myofiber from mouse tibialis anterior muscle electroporated with a GFP tagged DNAJB4-WT (green) (D) or DNAJB4-R25Q (green) (E) and then immunostained with an antibody to α-actinin (red). Scale bar is 10µM.

Article Snippet: Antibodies used were the following: anti-rabbit GAPDH (Cell Signaling, 2118), anti-mouse Desmin (Dako, M0760), anti-rabbit DNAJB6 (Abcam, ab198995), anti-mouse HspA1 (Enzo, ADI-SPA-810), anti-rabbit α-actinin (Abcam, ab68167), anti-rabbit Myotilin (Abcam, ab78492), anti-rabbit Synemin (Bioss, bs-8555R), anti-rabbit αβ-crystallin (Enzo, ADI-SPA-223), anti-rabbit DNAJB4 (proteintech, 13064-1-AP) and anti-human DNAJB4 (Santa Cruz, sc-100711).

Techniques: Western Blot, Control, Stable Transfection, Expressing, Confocal Microscopy, Imaging

Journal: bioRxiv

Article Title: Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure

doi: 10.1101/2022.07.31.502226

Figure Lengend Snippet: A) Rendering of DNAJB4 structure denoting the C-terminal, G/F and J domains with and enlargement of the J-domain showing Helix I-IV. Red star marks the R25 residue, green stars denote J domain residues (A50 and E54) and G/F domain residues (F89/F91/F93/N95/P96/D98/F100) mutated in DNAJB6 associated myopathy. Alignment of Helix II (underlined) from DNAJB4, DNAJB1, DNAJB2, DNAJB6 and Sis1. The R25 (DNAJB4/DNAJB1) and the R27 (Sis1) residue is in red. B) Yeast spottings from colonies that delete Sis1 and then complement with empty vector (EV), DNAJB-WT, DNAJB1-R25Q, Sis1-WT or Sis1-R27Q when plated on FOA media (left spottings). Spottings on full media (YPD) are on the right. C) Representative images of Hela cells transfected with GFP-DNAJB4-WT (green), or GFP-DNAJB4-R25Q (green) and mCherry-TDP-43 (red) one hour post heat shock. D) HeLa cells were co-transfected with GFP, GFP-DNAJB4-WT, or GFP-DNAJB4-R25Q and mCherry-TDP-43. The percentage of cells with TDP-43 nuclear inclusions at baseline (0H), post-heat shock (1H) or following heat shock recovery (1+3H) are represented graphically. E) HeLa cells were co-transfected with GFP, GFP-DNAJB4-WT, or GFP-DNAJB4-R25Q and mCherry-TDP-43 transfected with the indicated constructs, subjected to heat shock at 42°C for 1 hour, and the percentage of ethidium homodimer-1–positive cells was quantitated. Data are presented as the percentage of cells found positive/dead.

Article Snippet: Antibodies used were the following: anti-rabbit GAPDH (Cell Signaling, 2118), anti-mouse Desmin (Dako, M0760), anti-rabbit DNAJB6 (Abcam, ab198995), anti-mouse HspA1 (Enzo, ADI-SPA-810), anti-rabbit α-actinin (Abcam, ab68167), anti-rabbit Myotilin (Abcam, ab78492), anti-rabbit Synemin (Bioss, bs-8555R), anti-rabbit αβ-crystallin (Enzo, ADI-SPA-223), anti-rabbit DNAJB4 (proteintech, 13064-1-AP) and anti-human DNAJB4 (Santa Cruz, sc-100711).

Techniques: Residue, Plasmid Preparation, Transfection, Construct

Journal: bioRxiv

Article Title: Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure

doi: 10.1101/2022.07.31.502226

Figure Lengend Snippet: A) Lysates from skeletal muscle and tissues indicated from DNAJB4 homozygous knockout mice or control littermates immunoblotted with an antibody to DNAJB4 or GAPDH. B) X-ray image highlighting the skeleton of 8 month old DNAJB4 knockout (B4KO) or C57 control. C) Quantitation of the kyphotic index from 4 and 8 month old control or DNAJB4 knockout (B4KO) mice. D) Mean holding impulse on an inverted screen for 4 and 8 month old C57 control and DNAJB4 KO mice. E) Peak force forelimb grip strength testing for 4- and 8-month-old C57 control and DNAJB4 KO mice. F-G) Weight of indicated isolated muscles (tibialis anterior (TA), gastrocnemius (Gast) and quadriceps (Quad)) normalized to total body weight for 4- and 8-monthold C57 control and DNAJB4 KO mice.

Article Snippet: Antibodies used were the following: anti-rabbit GAPDH (Cell Signaling, 2118), anti-mouse Desmin (Dako, M0760), anti-rabbit DNAJB6 (Abcam, ab198995), anti-mouse HspA1 (Enzo, ADI-SPA-810), anti-rabbit α-actinin (Abcam, ab68167), anti-rabbit Myotilin (Abcam, ab78492), anti-rabbit Synemin (Bioss, bs-8555R), anti-rabbit αβ-crystallin (Enzo, ADI-SPA-223), anti-rabbit DNAJB4 (proteintech, 13064-1-AP) and anti-human DNAJB4 (Santa Cruz, sc-100711).

Techniques: Knock-Out, Control, Quantitation Assay, Isolation, Muscles

Journal: bioRxiv

Article Title: Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure

doi: 10.1101/2022.07.31.502226

Figure Lengend Snippet: A) H&E staining of quadriceps muscle from 8-month-old control or 4- and 8-month old DNAJB4 KO mouse muscle. Fibers with internal nuclei are denoted with black arrows. B) NADH staining of tibialis anterior muscle from 8-month-old control or 4- and 8-month-old DNAJB4 KO mouse muscle. Fibers with central clearings are denoted with black arrows. C) H&E and NADH staining of diaphragm muscle from 4-month-old control or 4-month-old DNAJB4KO mice. Note myofiber atrophy and decreased diaphragm thickness. White arrows denote central clearings on NADH. D) NADH staining of diaphragm muscle from 8-month-old control or 8-month-old DNAJB4KO mice. Note myofiber atrophy and decreased diaphragm thickness. Scale bars are 50µM.

Article Snippet: Antibodies used were the following: anti-rabbit GAPDH (Cell Signaling, 2118), anti-mouse Desmin (Dako, M0760), anti-rabbit DNAJB6 (Abcam, ab198995), anti-mouse HspA1 (Enzo, ADI-SPA-810), anti-rabbit α-actinin (Abcam, ab68167), anti-rabbit Myotilin (Abcam, ab78492), anti-rabbit Synemin (Bioss, bs-8555R), anti-rabbit αβ-crystallin (Enzo, ADI-SPA-223), anti-rabbit DNAJB4 (proteintech, 13064-1-AP) and anti-human DNAJB4 (Santa Cruz, sc-100711).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure

doi: 10.1101/2022.07.31.502226

Figure Lengend Snippet: A) Immunoblots of lysates from 4-month-old mouse tibialis anterior of control or DNAJB4 knockout mice with antibodies to DNAJB4, desmin (DES), myotilin (MYOT), α-actinin, synemin, HSPA1, CRYAB, DNAJB6 or GAPDH. B) Quantitation of immunoblotted proteins from TA muscle at 4 months. N=5/condition. *, **, *** indicate p values 0.01, 0.001 and 0.0001. C) Immunoblots of lysates from 8-month-old mouse tibialis anterior of control or DNAJB4 knockout mice with antibodies to DNAJB4, desmin (DES), myotilin (MYOT), α-actinin, synemin or GAPDH. D) Quantitation of immunoblotted proteins from TA muscle at 8 months. N=5/condition. *, **, *** indicate p values 0.01, 0.001 and 0.0001.

Article Snippet: Antibodies used were the following: anti-rabbit GAPDH (Cell Signaling, 2118), anti-mouse Desmin (Dako, M0760), anti-rabbit DNAJB6 (Abcam, ab198995), anti-mouse HspA1 (Enzo, ADI-SPA-810), anti-rabbit α-actinin (Abcam, ab68167), anti-rabbit Myotilin (Abcam, ab78492), anti-rabbit Synemin (Bioss, bs-8555R), anti-rabbit αβ-crystallin (Enzo, ADI-SPA-223), anti-rabbit DNAJB4 (proteintech, 13064-1-AP) and anti-human DNAJB4 (Santa Cruz, sc-100711).

Techniques: Western Blot, Control, Knock-Out, Quantitation Assay

Journal: bioRxiv

Article Title: Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure

doi: 10.1101/2022.07.31.502226

Figure Lengend Snippet: A-C) Control C2C12 or one of two different DNAJB4 knockout (B4KO) lines were placed in differentiation media for 5 days and then the differentiation index, number of nuclei/myotube and total number of myotubes was quantitated. D) Immunoblots of lysates from undifferentiated (0) or 5- and 10-days post differentiation of control or DNAJB4 knockout C2C12 cells (B4KO1 or B4KO2) with antibodies to DNAJB4, desmin, α-actinin or GAPDH. E) Immunoblots of lysates from differentiated (5 days) C2C12 or B4KO myotubes following detergent lysis and ultracentrifugation into a total (T) lysate, soluble (S) fraction and insoluble (I) fraction with antibodies to DNAJB6, desmin, CRYAB and myotilin. F) Immunofluorescent images of 5 day differentiated C2C12 or B4 KO myotubes using an antibody to desmin (green) and myotilin (red upper panels) or phalloidin (red lower panels). G) Graph of the percentage of fibers with desmin inclusions in control or DNAJB4 KO myotubes.

Article Snippet: Antibodies used were the following: anti-rabbit GAPDH (Cell Signaling, 2118), anti-mouse Desmin (Dako, M0760), anti-rabbit DNAJB6 (Abcam, ab198995), anti-mouse HspA1 (Enzo, ADI-SPA-810), anti-rabbit α-actinin (Abcam, ab68167), anti-rabbit Myotilin (Abcam, ab78492), anti-rabbit Synemin (Bioss, bs-8555R), anti-rabbit αβ-crystallin (Enzo, ADI-SPA-223), anti-rabbit DNAJB4 (proteintech, 13064-1-AP) and anti-human DNAJB4 (Santa Cruz, sc-100711).

Techniques: Control, Knock-Out, Western Blot, Lysis

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Chemical structure of UNC1666, with inhibition constant (K i ) of 0.16 nM for Mer (enzymatic IC 50 : 0.55 nM) and 0.67 nM for Flt3 (enzymatic IC 50 : 0.69 nM). (B) Chemical structure of UNC1653, which lacks significant activity against Mer (enzymatic IC 50 : 560 nM) and Flt3 (enzymatic IC 50 : 220 nM) and is used as a negative control in these studies. (C) Whole cell lysates from AML cell lines with known Flt3 mutation status were analyzed by immunoblot and demonstrate presence or absence of the Mer tyrosine kinase (above) and the Flt3 tyrosine kinase (middle). Actin is shown as an indicator of total protein (below).

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Inhibition, Activity Assay, Negative Control, Mutagenesis, Western Blot

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Mer was immunoprecipitated from AML cell lysates and phosphorylated ( p -Mer) and total Mer (~180 kDa) levels were assessed by immunoblot analysis. This representative blot of the Kasumi-1 cell line demonstrates decreased Mer phosphorylation after treatment with increasing doses of UNC1666. (B) Flt3 was immunoprecipitated from AML cell lysates and phosphorylated ( p -Flt3) and total Flt3 (130/160 kDa) levels were assessed by immunoblot analysis. This representative blot of the MV4;11 cell line demonstrates decreased Flt3 phosphorylation after treatment with increasing doses of UNC1666. (C) Inhibition of downstream signaling after administration of UNC1666 in a Mer expressing AML cell lines that does not express a Flt3-ITD mutation (Kasumi-1) compared with equivalent concentrations of vehicle (DMSO) or inactive control TKI UNC1653. Actin is shown as an indicator of total protein. (D) Downstream signaling after administration of UNC1666 in a Flt3-ITD AML cell line that does not express Mer (MV4;11). Representative blots from at least 3 independent experiments are shown. nM = nanomolar

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Inhibition, Expressing, Mutagenesis, Control

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: Mer pos or Flt3-ITD AML cell lines were treated with UNC1666, vehicle (DMSO), or inactive control TKI UNC1653 for 72 hours and then analyzed by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide to identify apoptotic and dead cells. (A) Representative flow cytometry profiles of Kasumi-1 cells are shown. The percentages of live (lower left quadrant), early apoptotic (lower right quadrant), and late apoptotic/dead cells (upper quadrants) are shown. (B) Graphic representation of flow cytometric analyses of apoptotic/dead cells. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, *** p < 0.001, NS = not significant. (C) Cells were treated as indicated for 72 hours, whole cell lysates were prepared and the indicated apoptotic proteins were assessed by immunoblot analysis. Actin is shown as a loading control.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Control, Flow Cytometry, Staining, Derivative Assay, Western Blot

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: Mer positive or Flt3-ITD AML cell lines were treated with UNC1666, vehicle (DMSO), or inactive control TKI UNC1653 for 72 hours, fixed with 100% ethanol and then analyzed by flow cytometry after staining with propidium iodide to identify stage of cell cycle. Graphic representation of cell cycle progress in Kasumi-1 and MV4;11 cells using ModFit analysis are shown. The percentages of cells in G2/M (light gray), S (white), and G1 (dark gray) phases are shown. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Control, Flow Cytometry, Staining, Derivative Assay

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Diagram of the replating assay. Cells were treated with UNC1666 or vehicle for 72 hours, then washed to remove any residual compound and equal numbers of viable cells (1.5 × 10 4 /ml) were replated in growth medium on Day 0. On Day 6 after replating, the number of viable cells was determined. (B) Graphic representation of the results of the replating assay in Mer or Flt3-ITD expressing cell lines demonstrating decreased rebound growth after treatment with UNC1666. Mean values and standard errors were derived from at least 3 independent experiments. (C) Colony-formation assays were performed using Mer or Flt3-ITD expressing AML cell lines. Cells were grown in soft agar with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle or negative control TKI UNC1653. Mean values and standard errors were derived from at least 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, NS = not significant.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Expressing, Derivative Assay, Negative Control

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Immunoblot analysis of Mer and Flt3 expression in lysates prepared from AML patient samples. (B) Flt3 mutation status of patient samples determined by molecular profiling. (C, D) Dose-dependent inhibition of Mer and Flt3 phosphorylation in response to treatment with UNC1666. AML blasts from patient sample #10510 (Mer positive, Flt3-ITD high allelic ratio) were treated with UNC1666 or vehicle for two hours. (C) Mer and Flt3 were immunoprecipitated from cell lysates and phosphorylated Mer ( p -Mer), total Mer (~180 kDa), phosphorylated Flt3 ( p -Flt3) and total Flt3 (130/160 kDa) were detected by immunoblot. (D) Phosphorylation of downstream signaling molecules was assessed by immunoblot after treatment with UNC1666 or vehicle.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Western Blot, Expressing, Mutagenesis, Inhibition, Phospho-proteomics, Immunoprecipitation

Journal: Oncotarget

Article Title: Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia

doi:

Figure Lengend Snippet: (A) Graphic representation of apoptosis and cell death in AML patient samples after treatment with UNC1666 or vehicle for 72 hours. Apoptotic and dead cells were determined by flow cytometry after staining with YO-PRO-1 iodide and propidium iodide. Values derived from each sample are shown. (B) Colony-forming assays were performed in methylcellulose with the indicated treatments. Graphic representation of reduced colony number after treatment with UNC1666, compared to vehicle. Mean values and standard errors derived from triplicate samples are shown. (C) Graphic representation of the effect of UNC1666 on normal cord blood colony forming potential. Mean values and standard errors were derived from 3 independent experiments. ** p < 0.01, NS = not significant.

Article Snippet: Additionally, UNC1666 inhibits Flt3 (MCE IC 50 0.69 nM; K i 0.67 nM) equipotently in enzymatic MCE assays.

Techniques: Flow Cytometry, Staining, Derivative Assay