Journal: Current Biology

Article Title: Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a ( Ink4a/Arf )

doi: 10.1016/j.cub.2017.09.007

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (clone 197G2) , Cell Signaling Technology , Cat# 4377; RRID: AB_331775.

Techniques: Recombinant, Produced, Reverse Transcription, Microarray, Labeling, DNA Purification, Methylation, TA Cloning, Sequencing, SYBR Green Assay, Software

Journal: bioRxiv

Article Title: Whole body regeneration deploys a rewired embryonic gene regulatory network logic

doi: 10.1101/658930

Figure Lengend Snippet: (A) Immunohistochemistry using and anti-pERK antibody revealing the activated/phosphorylated form of ERK (green) in uncut and regenerating polyps from 1 hpa to 144 hpa (counterstaining: DAPI). (B) Schematic representation of the various U0126 treatments that were applied during different windows of time during regeneration in the subsequent experiments. Polyps were fixed at the end of each treatments and analyzed. (C) Immunohistochemistry and Western blot (D) using and anti-pERK antibody (green) at various moments during regeneration in the absence or presence of the MEK inhibitor U0126 (Control condition: DMSO). In addition to anti-pERK, anti-tERK (total ERK) and anti-alpha-tubulin antibodies were used as controls for the western blot (D). (E) Phenotypic analysis resulting from UO126 treatments. The plot represents the regeneration step (x-axis) the treated polyps are in and the y-axis indicates the duration of U0126 treatments. 0.5 indicates the step following wound healing. The size of the dots and the blue shades represents the number of individuals per step and the percentage of polyps per treatment per step, respectively. (F) Diagram of results obtained from the compression/wound healing assays comparing UO126 treated polyps (red bars) during the first 10 hours of regeneration to DMSO controls (blue bars). (G) Confocal stacks of regenerating polyps counterstained with DAPI (cyan) focused at the mesenteries at 20 hpa after a 0-20 or 8-20 hpa treatment with UO126 and the dotplot shows the distribution of polyps between steps 0.5 and 1 according to the treatments. (H-I) Number of EdU+ cells (green) per polyps in the body wall epithelium (H) and the mesenteries (I), respectively. The x-axis shows the various 24 hours windows of treatment with UO126 (blue dots) or DMSO (red dots) and the y-axis show the number of EdU+ cells per polyp, represented by single dots (student t-test p.value <0.01).

Article Snippet: Then the nitrocellulose membrane was saturated with Salin buffer (Tris 0,24g/L, NaCl 1,63g/L, 5%cBSA, 0.5% Tween-20) and incubated 1 hour at room temperature with 1/2500 of primary antibody anti-pERK (Cat.#4377; Cell Signaling Technology) diluted saline buffer.

Techniques: Immunohistochemistry, Western Blot

Journal: bioRxiv

Article Title: RAB5c controls the assembly of non-canonical autophagy machinery to promote phagosome maturation and microbicidal function of macrophages

doi: 10.1101/2025.03.25.645097

Figure Lengend Snippet: RAW264.7 cells or BMDM were transduced with scrambled shRNA (sh_Ctrl) or Rab5c shRNA (sh_ Rab5c ). (A) Immunoblot analysis of VPS34-PI(3)K CIII complex on BSA-Bead- or IgG-Bead-containing phagosomes purified from RAW264.7 cells at 40 min after stimulation. Data shown is representative of 3-5 experiments performed independently. (B) Representative confocal images of RAW264.7-p40 phox -PX-Venus cells fed with Op-Zym for 25 min. The fluorescence intensity is displayed using the SMART LUT. *, phagosomes. Scale bar: 10 μm. (C) Quantification of time-lapse confocal microscopy analysis of p40 phox -PX-Venus at phagosomes containing Op-Zym in RAW264.7 cells. Fluorescence profile was normalized by the MFI at 0” for each phagosome. Squares are the mean and shaded areas are ± S.E.M. for 18 phagosomes. The area under the curve (AUC) was determined for each analyzed phagosome and statistical significance was calculated by unpaired Student′s t -test. Data shown is representative of 2 independent experiments (related to Video S3) . (D-E) Immunoblot analysis (D) and densitometric quantification (E) of LC3A/B-II in RAW246.7 cells non-stimulated or incubated with Op-Zym for 25 min; C8 (2 μM) for 25 min; DMXAA (50 μg/mL) for 1 h; PP242 (1 μM) for 2 h, as indicated. GAPDH was used as loading control. Bars are the mean values of 3 independent experiments (each indicated as a circle), and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t -test. (F) Immunoblot analysis of NOX2 components on IgG-Bead or BSA-Bead containing phagosomes purified from RAW264.7 cells 40 min after stimulation. Data shown are representative of 4-5 experiments performed independently. (G) Representative DIC images of formazan deposits in phagosomes of RAW264.7 cells or BDMD fed Op-Zym for 15 min. Arrowheads indicate Op-Zym-containing phagosomes positive (Red) or negative (Yellow) for formazan deposition. (H-I) Percentage of NBT + phagosomes containing Op-Zym in RAW264.7 cells (H) and BMDM (I). ( J-L) Representative confocal images (J) and MIF quantification of p-p47 phox immunolabeling (AF-647) at phagosomes in RAW264.7 cells fed Op-Zym for 15 min (K) or 40 min (L). (M-N) Representative confocal images (M) and MIF quantification (N) of p-ERK1/2 immunolabeling (AF-647) in RAW264.7 cells fed with Op-Zym for 15 min. Blue: nuclei (DAPI). Scale bar: 10 μm. (K, L, N) Gray objects are the MIF of each analyzed phagosome. Colored objects are the mean value for each independent experiment (n=3) and error bars are ± S.E.M. Statistical significance was calculated unpaired Student′s t -test between the indicated groups. (O) Percentage of NBT + phagosomes in RAW264.7 cells treated or not with ERK1/2 inhibitor (BVD-523; 100 μM) for 10 min and subsequently fed Op-Zym for 15 min. (H, I, O) Bars are the mean value of biological replicates (n=3-6, each indicated as a circle) and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t -test (H, I) or by one-way ANOVA and Tukey′s multiple comparisons (O) between the indicated groups. Data shown is representative of 3 (H, I) or 2 (O) experiments performed independently.

Article Snippet: Anti-RAB5c [Thermo Fisher Scientific, PA5-39408; IB, 1:2,000; Immunofluorescence (IF), 1:300], anti-GAPDH [Cell Signalling Technology (CST), Clone D16H11, 5174T, IB, 1:10,000], anti-β-actin (CST, Clone 8H10D10, 12262, IB, 1:5,000), anti-Microtubule-associated protein 1 light chain B (anti-MAPLC3B/LC3B; CST, 27759, IB, 1:1,000), anti-LC3 (LC3A/LC3B/LC3C; MBL Int., PM036, IF: 1:250), anti-RAB5a (CST, 2143, IB, 1:1,000), anti-RAB5b (Santa Cruz, Clone F-9, sc373725, IB, 1:1,000), anti-EEA1 (CST, Clone C45B10, 3288, IB, 1:1,000), anti-UNC93B (Abcam, 69497, IB, 1:3,000), anti-GM130 (MBL, Clone 5G8, M1793, IB, 1:500), anti-UVRAG (CST, Clone D2Q1Z, 13115, IB, 1:1,000), anti-Phosphatidylinositol 3-kinase catalytic subunit type 3 (anti-PIK3C3/VPS34; Clone D9A5, CST 4263, IB, 1:1,000), anti-Beclin1 (CST, 3738, IB, 1:1,000), anti-Rubicon (Clone D9F7, CST, 8465, IB, 1:1,000), anti-p-p47 phox (S328) (Abcam, 111855, IF, 1:50), anti-p47 phox (Abcam, Clone erp27205, 308256, IB, 1:1,000; IF, 1:250), anti-p-p40 phox (Thr514) (CST, 4311, IB, 1:1,000), anti-p22 phox (Abcam, 75941 IB, 1:1,000), anti-gp91 phox (BD Bioscience, Clone53, 611414, IB, 1:2,000), anti-ERK1/2 (CST, Clone 1375F, 4695, IF, 1:500), anti-p-ERK1/2(Thr202/204) (CST, Clone 197G2, 9106, IF, 1:100), anti-ATP6V0d1 (Abcam, Clone erp18320-38, IB, 1:1,000), anti-ATP6V1A (Abcam, Clone erp19270, 199326, IB, 1:4,000; IF, 1:250), anti-ATG16L1 (MBL, Clone 1F12, M150-3, IB, 1:1,000), anti-ATG5/12 (CST, Clone D5F5U, 12994, IB, 1:1,000), anti-LAMP1 (CST, Clone E5N9Z, #99437, IF, 1:150), Alexa Fluor 647 polyclonal goat anti-rabbit IgG (Thermo Fisher Scientific, A21244, IF: 1:750), Alexa Fluor 488 polyclonal donkey anti-mouse IgG (Thermo Fisher Scientific, A21202, IF: 1:750), Alexa Fluor 647 polyclonal donkey anti-mouse IgG (Thermo Fisher Scientific, A31571, IF: 1:750), HRP-conjugated anti-rabbit IgG (CST, 7074, IB: 1:5,000), and HRP-conjugated anti-mouse IgG (CST, 7076, IB: 1:5,000) were used as indicated in manufactures′ datasheets.

Techniques: Transduction, shRNA, Western Blot, Purification, Fluorescence, Confocal Microscopy, Incubation, Control, Immunolabeling