Journal: Cellular and molecular life sciences : CMLS

Article Title: Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

doi: 10.1007/s00018-023-04850-4

Figure Lengend Snippet: Fig. 6 Role of Sorcin in cell migration after EGF and IGF treatment. Wound-healing assay in H1299 cells cultured in serum-free medium (SF) or in the presence of EGF and IGF, in control experiments (siNC) and upon silencing of Sorcin (siSRI). Images were acquired at 0 h and 24 h after each single treatment. Quantification of wound-healing assay calculated as percentage of wound closure: ((At0 – At1)/ At0) × 100), where At0 is the initial wound area and At1 is the wound area n hours after the initial scratch. Error bars indicate means ± SEM. **p < 0.01, ***p < 0.001 and ****p < 0.0001 as determined by Student’s t test (n = 3). Scale bars, 200 µm

Article Snippet: Western blotting was performed loading 30 μg of lysates and using the following primary antibodies: rabbit monoclonal EGFR (1:1000 in 5%BSA-T-TBS solution) (D38B1, #4267, Cell Signaling), rabbit monoclonal Phospho-EGF Receptor (1:1000 in 5%BSA-T-TBS solution) (Tyr1068, #3777 Cell Signaling, Danvers, MA, USA), rabbit monoclonal p44/42 MAPK (Erk1/2) (1:1000 in 5%BSA-T-TBS solution) (137F5, #4695 Cell Signaling, Danvers, MA, USA), rabbit monoclonal Phospho-ERK1/ERK2 (P44/ P42 MAPK) (1:1000 in 5%BSA-T-TBS solution) (T202/ Y204 # MAB-94112 Immunological Science, Italy), rabbit polyclonal CHD2 (1:1000 in 5%BSA-T-TBS solution) (#ab182013, Abcam), mouse monoclonal SLUG (A-7) (1:200 in 5%BSA-T-TBS solution) (#sc-166476 Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal SNAI1 (G-7) (1:200 in 5%BSA-T-TBS solution) (#sc-271977 Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal Sorcin (39-M) (1:200 in 5%BSA-T-TBS solution) (#sc-100859 Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal GAPDH (1:2000 in 5%BSA-T-TBS solution) (#TA802519, OriGene Technologies, Rockville, USA), mouse monoclonal α-Tubulin B-5–1-2 (1:3000 in 5%BSA-T-TBS solution) (#T5168, Sigma-Aldrich, Milan, Italy).

Techniques: Migration, Wound Healing Assay, Cell Culture, Control

Journal: bioRxiv

Article Title: Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells

doi: 10.1101/2022.03.03.482327

Figure Lengend Snippet: A) Graphical timeline illustrating changes in the expression of Ca 2+ related genes during OHC development (key on right). At P0, APV (red) expression is abundant . Sorcin (orange) expression is also observed. By P3, OCM (green) protein expression is upregulated, while APV expression is downregulated . B) Maximum Intensity Projection of mid-modiolar organ of Corti preparation from adult mouse (3 mo.). Sample was stained with OCM (white), prestin (red), phalloidin (green) and DAPI (blue). Inset shows details of OHC sub-nuclear region. C) Relative Ocm transcript levels measured via qRT-PCR in P0 and P3 Ocm +/- and Ocm -/- mice. Bars represent mean transcript levels (normalized to B2M , a housekeeping gene) and are plotted relative to P0 Ocm +/- . Error bars represent S.E.M. Significance ( t test) is denoted as: **p=0.005 . D) Confocal images of cochlear spirals harvested from P0 and P3 Ocm +/- and Ocm -/- mice. OCM (white), phalloidin (green) and DAPI (blue) are shown. Scale bar = 10μm. Maximum Intensity Projections are composed of 2 × 1 μm slices. 63x magnification.

Article Snippet: Samples were stained with antibodies to OCM (Santa Cruz sc-7446, 1:200), APV (SWANT PVG-213, 1:200), and sorcin (Invitrogen PA5-64975, 1:200).

Techniques: Expressing, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells

doi: 10.1101/2022.03.03.482327

Figure Lengend Snippet: A-B) qRT-PCR was used to determine relative transcript levels in cochlear spirals harvested from P0 and P3 Ocm +/- (white) and Ocm -/- (red) mice. Mean values of relative normalized expression of A) Apv and B) Sri are represented by a bar graph. Mean transcript levels are plotted relative to P0 Ocm +/- mean values. Error bars represent S.E.M. Significance ( t test) is denoted as: ***p<0.001 . Of note, when a one-tailed t test was used to compare mean values of Apv mRNA abundance between P0 Ocm +/- vs Ocm -/- , there is a significant difference ( p=0.048 ) C-D) Confocal images of cochlear spirals microdissected from P0 and P3 Ocm +/- and Ocm -/- mice. C) APV (white) and D) sorcin (red) were detected at P0 and P3 in both Ocm +/- and Ocm -/- mice. Phalloidin and DAPI (not shown) were used to outline OHCs and denote subcellular location. Maximum Intensity Projections of 1 μm slices starting from the cuticular plate and ending in the nucleus are shown here. Scale bar = 10 μm.

Article Snippet: Samples were stained with antibodies to OCM (Santa Cruz sc-7446, 1:200), APV (SWANT PVG-213, 1:200), and sorcin (Invitrogen PA5-64975, 1:200).

Techniques: Quantitative RT-PCR, Expressing, One-tailed Test

Journal: bioRxiv

Article Title: Oncomodulin (OCM) uniquely regulates calcium signaling in neonatal cochlear outer hair cells

doi: 10.1101/2022.03.03.482327

Figure Lengend Snippet: HEK293T cells were transiently transfected with fluorescently tagged CaBPs. A) Ocm mRNA is not endogenously detected in HEK293T cells. G3PDH and Ocm cDNA generated via RT-PCR from cell lysates of transfected ( Ocm -CFP) and untransfected HEK293T cells. Wells labeled “-” are no transcript controls. B) HEK293T cells expressing fluorescently tagged OCM or control (untransfected) were incubated with Fluo-4 and stimulated with ATP. Changes in fluorescence of single cells are shown in grey. The solid black line indicates mean ΔF/F0 for that group. Dotted red lines represent control experiments including CPA, a SERCA inhibitor. C) Representative plots of ΔF/F0 upon stimulation with ionomycin. Changes in fluorescence of single cells are shown in grey. Solid black line indicates mean ΔF/F0 for that group. Dashed green line marks 30 s post stimulation. D) Fluorescent proteins (red): mCh-only, Ocm -mCh, Apv -mCh (not shown), Sri -mCh (not shown) were incubated with Fluo-4 (green) and stimulated with ionomycin. Shown are still images before (0 s) and 30 s post-stimulation with ionomycin. Scale bar = 50 μm. E) Violin plots of ΔF/F0 30 seconds post stimulation with ionomycin for HEK293T cells positive for: mCh (white), OCM (pink), APV (teal), and sorcin (purple). Solid lines represent medians. Dashed lines represent quartiles. n≥100 cells per group. Significance ( one-way ANOVA ) denoted as: * p=0.02 and *** p<0.001

Article Snippet: Samples were stained with antibodies to OCM (Santa Cruz sc-7446, 1:200), APV (SWANT PVG-213, 1:200), and sorcin (Invitrogen PA5-64975, 1:200).

Techniques: Transfection, Generated, Reverse Transcription Polymerase Chain Reaction, Labeling, Expressing, Incubation, Fluorescence