s2 cells  (Thermo Fisher)

Journal: The Journal of Cell Biology

Article Title: Drosophila Abi maintains blood cell homeostasis by promoting clathrin-mediated endocytosis of Notch

doi: 10.1083/jcb.202505091

Figure Lengend Snippet: Abi acts through WASp to regulate CME and crystal cell development. (A) Single confocal slices of S2 cells pretreated with 100 μM dynasore for 30 min prior to immunofluorescence analysis using anti-Abi (green) and anti-Chc (cyan) antibodies together with an anti-WASp or anti-SCAR (pseudocolored magenta) antibody. Arrowheads indicate Abi + punctae colocalizing with WASp and Chc. Arrows indicate Abi + punctae colocalizing with SCAR but not Chc. (B) Single confocal slices of primary hemocytes from WT, abi 5 /Df , UAS-HA-abi Δ30–65 /+; HmlΔ-GAL4 , abi 5 /+, Df ( abi 5 /Df , HmlΔ > abi Δ30–65 ), HmlΔ-GAL4 , abi 5 / UAS-HA-abi W452K , Df ( abi 5 /Df , HmlΔ > abi W452K ), HmlΔ-GAL4 /+, HmlΔ-GAL4 / UAS-SCAR RNAi ( HmlΔ > SCAR RNAi ), and HmlΔ-GAL4 / UAS-WASp RNAi ( HmlΔ > WASp RNAi ) late-third instar larvae. Hemocytes were pulsed with Alexa Fluor 555-mBSA (red) for 5 min, chased for 5 min, and stained with DAPI (blue). (C) Quantification of the ratio of mean Alexa Fluor 555-mBSA to DAPI fluorescence intensities. HmlΔ-GAL4 , abi 5 / UAS-HA-abi , Df ( abi 5 /Df , HmlΔ > abi ) was also analyzed. Values are presented as percentages of WT or HmlΔ-GAL4 /+. n = 30 hemocytes. (D) Bright-field images of heated (70°C, 10 min) late-third instar larvae of the indicated genotypes. Dorsal views of the two most posterior segments (A7 and A8). (E) Number of heat-blackened crystal cells in the A7 and A8 segments. n = 12 larvae. (F–I) Transheterozygous interactions between abi and WASp . (F) Single confocal slices of primary hemocytes from late-third instar larvae of the indicated genotypes. Hemocytes were pulsed with Alexa Fluor 555-mBSA for 5 min and chased for 5 min. (G) Quantification of the ratio of mean Alexa Fluor 555-mBSA to DAPI fluorescence intensities. Values are presented as percentages of WT. n = 30 hemocytes. (H) Bright-field images of heated (70°C, 10 min) late-third instar larvae of the indicated genotypes. (I) Number of heat-blackened crystal cells in the A7 and A8 segments. n = 12 larvae. Data represent the mean ± SEM. Comparisons are with WT or HmlΔ-GAL4 /+ (***P < 0.001). Statistical analyses were performed using a one-way ANOVA with the Tukey–Kramer post hoc test. The dashed lines define cell boundaries. Scale bars: 10 μm (A); 5 μm (B and F); 200 μm (D and H).

Article Snippet: S2 cells were transfected in serum-free medium using Cellfectin (Invitrogen), following the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Nucleic Acids Research

Article Title: Phosphorylation of Barrier-to-Autointegration Factor (BAF) regulates anchoring of centromeric heterochromatin to the nuclear envelope (NE)

doi: 10.1093/nar/gkag021

Figure Lengend Snippet: Impaired BAF phosphorylation reinforces heterochromatin anchoring. ( A ) Immunostaining with αLamin antibodies (in cyan) in stable S2 cells co-expressing mCherry::HP1a (in red) and either N-BAF, C-BAF, or N-BAF T4A/S5A . mCherry::HP1a signal is direct fluorescence. Scale bars are 2 μm. ( B ) Quantification of the results shown in panel (A). The proportion of HP1a foci in the peripheral, intermediate, and central nuclear regions is shown for cells expressing N-BAF, C-BAF, or N-BAF T4A/S5A (see “Materials and methods” section for details). As a control, a similar quantification was performed in S2 cells after co-immunostaining with αLamin and αHP1a antibodies. Results are the sum of two independent experiments. ( N = 91 for N-BAF, 93 for C-BAF, and 111 for N-BAF T4A/S5A ; P -values with respect to control: ns > 0.05, ***** < 0.00001; two-tailed paired Student’s t -test). ( C ) Quantification of the results shown in panel (A). The number of HP1a foci per nucleus is shown for cells expressing N-BAF, C-BAF, or N-BAF T4A/S5A . Results are the sum of two independent experiments. Error bars are SD. ( N = 35 for N-BAF, 34 for C-BAF, and 33 for N-BAF T4A/S5A ; P -values with respect to control: ***** < .00001; two-tailed paired Student’s t -test). ( D ) ChIP-seq genomic profiling of endogenous BAF, N-BAF, C-BAF, and N-BAF T4A/S5A . For endogenous BAF, ChIP was performed with αBAF antibodies in Drosophila S2 cells. For N-BAF, C-BAF, and N-BAF T4A/S5A , ChIPs were performed with αGFP antibodies in stable S2 cells expressing the corresponding BAF forms. The IP/Input coverage ratio is presented for two representative genomic regions and compared to Lamin (GEO GSM509086 ) . LADs are indicated in gray. The Spearman correlation coefficients respect to Lamin for the whole genome are also indicated.

Article Snippet: To obtain stable cell lines, Drosophila S2 cells were grown under standard conditions [25°C in Schneider’s medium (Sigma) supplemented with 10% FBS (Gibco), 100 mg/ml Streptomycin, and 100 mg/ml Penicillin] and transfected with the appropriate constructs by the calcium phosphate method as described in [ ].

Techniques: Phospho-proteomics, Immunostaining, Expressing, Fluorescence, Control, Two Tailed Test, ChIP-sequencing