human melanoma g361 cells  (ATCC)

Journal: Annals of dermatology

Article Title: Overexpression of Dock180 and Elmo1 in Melanoma is Associated with Cell Survival and Migration.

doi: 10.5021/ad.23.023

Figure Lengend Snippet: Fig. 2. Apoptotic effects following knockdown of Dock180 and Elmo1 in G361 and SK-MEL-2 cells. Cells were treated with Dock180- or Elmo1-specific small interfering RNA (siRNA). (A) The percentage of cell viability was measured by MTT assay as mean±standard devia- tions for three independent experiments. (B) Phase contrast images of G361 and SK-MEL-2 cells. (C) In DAPI staining, both chromatin con- densation and nuclear fragmentation were increased in G361 and SK-MEL-2 cells following treatment of si-Dock180 or si-Elmo1. si-Ctrl: siRNA control, si-Dock180: siRNA targeting Dock180, si-Elmo1: siRNA targeting Elmo1. *p<0.05 compared to untreated controls.

Article Snippet: The human melanoma cell lines G361 and SK-MEL-2 were purchased from the American Type Culture Collection (CRL- 1424 and HTB-68TM; ATCC).

Techniques: Knockdown, Small Interfering RNA, MTT Assay, Staining, Control

Journal: Annals of dermatology

Article Title: Overexpression of Dock180 and Elmo1 in Melanoma is Associated with Cell Survival and Migration.

doi: 10.5021/ad.23.023

Figure Lengend Snippet: Fig. 4. Inhibition of Dock180 and Elmo1 changes cell cycles and apoptosis of G361 and SK-MEL-2 cells. (A) Cell distribution at G0/G1, S, and G2/M phases was analyzed at 48 hours of incubation time using flow cytometry after staining with propidium iodide (20 μg/ml). (B) The percentage of apoptotic cells after Annexin V-PE binding was analyzed at 48 hours of incubation time using a Muse cell analyzer. The quantitative data were shown as mean±standard deviations for three independent experiments. si-Ctrl: siRNA control, si-Dock180: siRNA targeting Dock180, si-Elmo1: siRNA targeting Elmo1. *p<0.05 compared to untreated controls.

Article Snippet: The human melanoma cell lines G361 and SK-MEL-2 were purchased from the American Type Culture Collection (CRL- 1424 and HTB-68TM; ATCC).

Techniques: Inhibition, Incubation, Flow Cytometry, Staining, Binding Assay, Control

Journal: Annals of dermatology

Article Title: Overexpression of Dock180 and Elmo1 in Melanoma is Associated with Cell Survival and Migration.

doi: 10.5021/ad.23.023

Figure Lengend Snippet: Fig. 5. Inhibition of migration and colony formation in G361 and SK-MEL-2 cells by knockdown of Dock180 and Elmo1. (A) Transfec- tion with Dock180- or Elmo1-specific small interfering RNA (siRNA) induced the inhibition of melanoma cell migration. At 24 hours post-transfection, the cells were grown to near confluence, and then wounded by dragging a 20 µl pipette tip through the monolayer. Cell migration images were captured soon after the wound was introduced (0 hour) and at a designated time (48 hours after wounding) under a microscope. (B) In 2 weeks after transfection with Dock180- or Elmo1-specific siRNA, colony formation of the G361 cells was suppressed. Immunoblot analysis of Dock180, Elmo1, Rac1 and phosphorylation of ERK and AKT in G361 and SK-MEL-2 cells treated with Dock180- or Elmo1-specific siRNA. si-Ctrl: siRNA control, si-Dock180: siRNA targeting Dock180, si-Elmo1: siRNA targeting Elmo1. *p<0.05 compared to untreated controls.

Article Snippet: The human melanoma cell lines G361 and SK-MEL-2 were purchased from the American Type Culture Collection (CRL- 1424 and HTB-68TM; ATCC).

Techniques: Inhibition, Migration, Knockdown, Small Interfering RNA, Transfection, Transferring, Microscopy, Western Blot, Phospho-proteomics, Control

Journal: Communications biology

Article Title: The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

doi: 10.1038/s42003-023-05236-9

Figure Lengend Snippet: Fig. 4 Increase of WIPI1 protein enhances autophagic flux. a Immunoblot analysis of LC3B lipidation in U2OS cells after transfection with control plasmids (9E10) or plasmids encoding 9E10-tagged WIPI1. U2OS cells were transfected with the indicated amounts of plasmids for 48 h before protein extraction and immunoblotting against LC3B, 9E10-tagged WIPI1 or GAPDH, n = 3. Additional immunoblots provided in Supplementary Fig. 5a. b U2OS Cas9 control or U2OS WIPI1 KO cells were transfected with control plasmids (9E10) or plasmids encoding 9E10-tagged WIPI1 for 48 h, followed by treatment with bafilomycin A1 (BafA1) in fed conditions for 3 h. Immunoblotting was conducted against LC3B, 9E10 and tubulin (n = 6, mean ± SD, Two-way ANOVA with Tukey’s multiple comparisons test). WIPI1 deficiency control Taqman qPCR is presented in Supplementary Fig. 6b, and representative Western blots in Supplementary Fig. 6c. c U2OS cells stably expressing GFP-WIPI2 were transfected with control plasmids or plasmids encoding mCherry-tagged WIPI1 for 48 h. The numbers of GFP-WIPI2 puncta cells were assessed by fluorescence microscopy in transfected cells. Welch’s t test, mean ± SD, up to 1272 analysed cells from n = 4 in duplicates. d U2OS Cas9 control or U2-OS WIPI1-KO cells were seeded into 96-well glass bottom plates and transfected with siABL1/2, siDDR1 or nontargeting siRNA (siControl) for 48 h, followed by treatment with either DMEM/FBS (fed) or EBSS (starved) for 3 h. After fixation, cells were stained with DAPI and anti-WIPI2/AF488. By automated confocal LSM, 20 images per well were acquired and between 621 to 2563 single cells (from n = 3) subjected to automated CellProfiler-based image analysis (threshold-based puncta segmentation). For statistical analysis, a two-way ANOVA with Tukey’s multiple comparisons test was performed (mean ± SD). e G361 cells were transfected with plasmids encoding GFP or GFP-WIPI1 and were fed or starved for 3 h in the presence or absence of bafilomycin A1, followed by anti-WIPI2/AF546 immunofluorescence staining. Confocal LSM stacks were acquired, and the numbers of WIPI2 puncta-positive cells per acquired image (individual data points represent the result derived from each image) were counted (left panel: two-way ANOVA with Tukey’s post-hoc test, mean ± SD, up to 215 single cells from n = 3 for each condition). Indicative of the presence of overexpressed GFP-WIPI1 are elongated, perinuclear autophagic membranes found to colocalize with WIPI2 (right panel: Scale bar: 5 μm, extended image presentation in Supplementary Fig. 5b). Supplementary material is available (Supplementary Fig. 6d; Supplementary Data 1). P values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant.

Article Snippet: Human U2OS osteosarcoma cells (ATCC, HTB-96) or human G361 malignant melanoma cells (ATCC; CRL-1424) were cultured in Dulbecco’s modified Eagle medium (DMEM) GlutaMAX (Life Technologies, 31966)/10% fetal bovine serum (Life Technologies, 10270-106) supplemented with 100 U/ml penicillin/100mg/ml streptomycin (Life Technologies, 15140-122) at 37 °C and 5% CO2.

Techniques: Western Blot, Transfection, Control, Protein Extraction, Stable Transfection, Expressing, Microscopy, Staining, Derivative Assay