EMBO/soda-nel huggingface dataset:
Glioma-derived cancer stem cells are hypersensitive to proteasomal inhibition (2016)
Figure 1

Figure 1. Validation of the in vitro culture model of GSCs.

(A) GSCs contain the higher levels of stemness markers (Nestin, SOX2, Musashi-1, NANOG, and OCT4) compared with their DFCs. Total RNAs from GSCs and DFCs were subjected to real-time PCR. The average relative amounts of GSCs were normalized with those of DFCs. Shown are means +S.D. of the normalized mRNA levels of GSCs obtained from the three independent experiments. (B) Immunoblotting analysis of markers for stemness (SOX2, Nestin and Musashi-1) and differentiation (GFAP and TUJ1 (Neuron-specific class III beta-tubulin)) in GSCs in comparison with GSC-derived cells at different passage numbers (P1-P7). (C) Measurement of the cell doubling time. Approximately 3×104 cells for each of XO8 GSCs and XO8 DFCs were seeded in a 60 mm culture dish and counted every day. The cells were treated with trypsin-EDTA, and the number of viable cells was counted with a hemocytometer. The doubling time was calculated from the cell growth curve over 5 days. Shown are means ±S.D. obtained from three independent experiments. (D) FACS analysis of CD133 and GFAP using X08 GSCs and DFCs. Data are from representative experiments repeated at least three times. (E) Immunoblotting analysis of CD133 and GFAP in GSCs in comparison with DFCs. (F) Quantitation of (G). (G) Equal numbers of XO8 GSCs and DFCs were injected orthotropically into mouse brains. After six weeks, brains were harvested and tumorigenic potential was assessed by evaluating tumor formation. Representative microphotographs of the brain sections of mice injected with XO8 GSCs and XO8 DFC controls.
Glioma-derived cancer stem cells are hypersensitive to proteasomal inhibition (2016)
Figure 2

Figure 2. GSCs are hypersensitive to PIs compared with their DFCs.

Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide (A), cisplatin (B), adriamycin (C), taxol (D), TRAIL (E), bafilomycin A1 (F), thapsigargin (G), and MG132 (H). Means ±S.D. from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO. (I) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs. Cell viability was measured using trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. (*P < 0.05). (J) XO8 GSCs were treated with MG132 were cultured in 0.3% soft-agar as described in Materials and Methods. The colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting. Bars, means +S.D. from three independent experiments.
Glioma-derived cancer stem cells are hypersensitive to proteasomal inhibition (2016)
Figure 4

Figure 4. The stemness of GSCs correlates to increased ubiquitination, and PIs induce GSC-specific apoptosis.

(A) Immunoblotting of XO8 GSCs and DFCs. (B) Immunoblotting analysis of Ub in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (C) Similar to (B) except that XO6 GSCs were used. (D) Immunoblotting analysis of ubiquitin ligases and proteasomal subunits in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (E) Similar to (C) except that XO6 GSCs were used. (F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039; (G), 0.0052; (H), 0.0009, (I) DFC, 0.00032; (I)-HEK293, 0.0039; (I)-HeLa: 0.00051. (J) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy. Scale bar, 100 μM. (K) Cells from (J) were subjected to flow cytometry analysis. Annexin V-positive cell numbers were obtained from three independent experiments and normalized to those of controls cells treated with DMSO. Bars, means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: 6 hrs, 0.0012; 17 hrs, 0.0041. (L) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers. (M and N) Similar to (L) except that XO6 and XO10 cells were used.
Glioma-derived cancer stem cells are hypersensitive to proteasomal inhibition (2016)
Figure 5

Figure 5. Analysis of the mRNA expression of proteasomal subunits and the NF-κB pathway in GSCs.

MG132 at low nM induces apoptosis in GSCs through the JNK mitogen-activated protein kinase (MAPK) pathway. (A) Real-time PCR analysis of the proteasomal subunits PSMB5, PSMB6, and PSMB7 in XO8 GSCs and XO8 DFCs. Shown are means +S.D. values from the three independent experiments following normalization to those of GSCs. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.053, PSMB6: 0.034, PSMB7: 0.067. (B) Similar to (A) except that the cells were treated with 50 nM MG132 for 17 hrs. Shown are means +S.D. values from the three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.022, PSMB6: 0.08, PSMB7: 0.603. (C) Immunoblotting analysis of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 and 12. Note that part of Figure 5C is also shown as the left panel of Figure 4A. (D) Same as (C) except that the cells were treated with MG132 for 17 hrs. (E) Immunoblotting analysis of the NF-κB pathway in XO8 GSCs and XO8 DFCs. (F) Fractionation analysis of NF-κB in XO8 GSCs and XO8 DFCs treated with 50 nM MG132 for 17 hrs. PARP1 and tubulin were used as markers for the nucleus and cytoplasm, respectively. (G) Immunoblotting analysis of JNK and p38. (H) Cell viability assay was performed as in Fig. 2 with cells treated with MG132 alone or in combination with SB203580. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value = 0.724, 0.502, 0.439 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.1 μM MG132, and 0.760, 0.683, 0.498 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.5 μM MG132. (I) Experimental conditions were similar to (H) except that SP600129 was used. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. * P value = 0.009, 0.002, 0.07 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.1 μM MG132, and 0.018, 0.025, 0.09 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.5 μM MG132. (J) Immunoblotting analysis of XO8 GSCs treated with 50 nM MG132 alone or in combination with the JNK inhibitor SP600129.
Glioma-derived cancer stem cells are hypersensitive to proteasomal inhibition (2016)
Figure 6

Figure 6. The expressions of ER stress-associated proapoptotic genes is selectively induced by MG132 in GSCs.

(A and B) XO8 GSCs and XO8 DFCs were treated with 50 nM MG132 for indicated hours. The expression of genes was assessed by real-time PCR using total RNA extracted from MG132-treated cells. Mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. (C) XO8 GSCs and XO8 DFCs treated with 50 nM MG132 as described in (A) were subjected to immunoblotting. (D) XO8 GSCs were treated with MG132, salinosporamide A, epoxomicin, or bortezomib for 24 hrs. Cell lysates were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP, anti-ATF3, anti-ATF3, and anti-BiP antibodies. (E) XO8 cells were treated with 50 nM MG132 in the absence or presence of 5μM actinomycin D (ActD) for 6 h. The level of ATF3 mRNA was measured using semi-quantitative real-time PCR in comparison with β-actin.
Glioma-derived cancer stem cells are hypersensitive to proteasomal inhibition (2016)
Figure 7

Figure 7. ATF3 and NOXA mediate low dose PI-induced apoptosis in GSCs.

(A) HEK293 cells were transfected with ATF3 shRNA or vector (pLKO1), treated with 50 nM MG132, and then subjected to immunoblotting. (B) Individualized XO8 GSCs were transduced with lentiviral shRNAs against ATF3 or control as described in Materials and Methods. After four days, individualized GSCs were treated with 50 nM MG132, and subjected to immunoblotting. (C) XO8 GSCs were transfected with control or CHOP siRNA for 48 hrs and then treated with 50 nM MG132 for 24 hrs. Cell viability was determined using the Trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used. (D) Immunoblotting analysis of (C). The cells were collected, lysed, and subjected to immunoblot analysis with indicated antibodies. (E) Wild-type and CHOP-/- MEF cells were treated with MG132 for 24 hrs. Cell viability was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from three separate experiments (*P < 0.05, n=3). For statistical analysis, student t-test (two-sided, one type) were used. (F) Cell lysates obtained in (E) were subjected to immunoblot analysis with anti-PARP-1, anti-CHOP or anti-ATF3 antibody. (G) The expression of NOXA was assessed with real-time PCR using total RNA extracted from XO8 GSCs treated with 50 nM MG132 alone or in combination with each inhibitor of ER stress-driven apoptosis (salubrinal; 15 µM, STK064652; 20 µM, STK047915; 20 µM). The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: salubrinal, 0.0003; STK064650, 0.0036; STK047915, 0.0019. (H) Similar to (C) except that XO8 GSC transduced with lentiviral shRNAs were treated with MG132. The mean +S.D. values from the three independent experiments are shown after normalization to that of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) was used. * P value: #1, 0.026; #5, 0.0033; #6, 0.0482. (I) XO8 GSCs were treated with 20 nM MG132 alone or in combination with each inhibitor as described in (C), and subjected to immunoblotting. Sal.: salubrinal.
Acetylcholine-synthesizing macrophages in subcutaneous fat are regulated by β2-adrenergic signaling (2021)
Figure 1

Figure 1. Acetylcholine-synthesizing macrophages reside in subcutaneous fat.

A - Three-dimensional imaging of WT and ChAT-eGFP IWAT using the Adipo-Clear method (Chi et al., 2018) and lightsheet fluorescence microscopy. Whole IWAT was stained with Alexa Fluor 488-conjugated anti-GFP antibody to visualize ChAT-eGFP-expressing cells. High-magnification sections are shown to the right of each sample. Scale bars: 100 μm.

B - UMAP plot displaying the profile of ChAT-eGFP+ cells from IWAT, analyzed by flow cytometry and combined from four biological replicates. ChAT-eGFP+ cell types are color‑coded with accompanying labels, and the percentage breakdown of ChAT-eGFP+ cells is featured in a bar chart to the right. Related to Table EV1. MΦ: macrophages.

C - Left: Percentage of total live cells expressing ChAT-eGFP derived from IWAT (including inguinal lymph node; n = 7), IWAT alone (n = 8) and lymph node alone (n = 8). Right: Proportion of ChAT-eGFP+ cells comprised by T cells, B cells and MΦ in each tissue type.

D - Total abundance of ChAT-eGFP+ CD45+ hematopoietic cells in IWAT, VWAT and BAT SVF isolated from ChAT-eGFP mice housed at room temperature (RT) or exposed to 4°C (CE) for 4 h (n = 4).

E - LC/MS-MS traces showing acetylcholine (Ach) levels in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT compared to an internal control (d4-Ach).

F - Left: relative mRNA expression of Chat in ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 9). Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: quantification by LC/MS-MS of Ach concentration in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 6).

G - Percentage of total IWAT SVF cells expressing ChAT-eGFP in male (n = 6) and female (n = 9) ChAT-eGFP mice housed at RT or CE (4 h).

H - Percentage of total IWAT T cells, B cells and MΦ that express ChAT-eGFP, from male (n = 6) and female (n = 5 for T cells and B cells; n = 8 for MΦ) mice housed at RT or CE (4 h).

I - Left: MΦ as a percentage of all ChAT-eGFP+ cells in IWAT at RT and 4 h CE (n = 15). Middle: Total number of ChAT-eGFP+ MΦ at RT and 4 h CE (n = 15). Right: ChAT-eGFP median fluorescence intensity (MFI) for ChAT-eGFP+ MΦ at RT and 4 h CE (n = 15).

J - MFI for Ki67 in ChAT-eGFP+ MΦ from IWAT at RT and 4 h CE (n = 6).

K - Schematic depicting the experimental strategy for transcriptomic profiling of ChAT-eGFP+ and ChAT-eGFP- MΦ from IWAT of mice housed at 4oC for 4 h.

L - Relative expression (pseudocounts) heatmap of genes relevant to acetylcholine signaling in ChAT-eGFP- (n = 4) and ChAT-eGFP+ (n = 3) MΦ.

M - Biological pathway analysis of significantly enriched genes in ChAT-eGFP+ MΦ.

Data information: In (C), data are presented as mean ± SEM and the letters "a", "b" and "c" indicate P < 0.05 between groups (one-way ANOVA). In (D and F-J), data are presented as mean ± SEM where *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed Student's t-test).
Acetylcholine-synthesizing macrophages in subcutaneous fat are regulated by β2-adrenergic signaling (2021)
Figure 2

Figure 2. Loss of ChAT in macrophages compromises the adaptive thermogenic capacity of subcutaneous fat.

A - Cell-specific deletion of ChAT was achieved by crossing ChATfl/fl mice with LysM-Cre (MΦ), Cd4-Cre (T cells) or Mb1-Cre (B cells) mice.

B - ChAT deletion was confirmed in MΦ, CD4+ and CD8+ T cells, and B cells sorted from IWAT of ChATfl/fl;LysM-Cre (n = 3), ChATfl/fl;Cd4-Cre (n = 4 for ChATfl/fl, n = 3 for Cre for CD4, n = 4 for CD8) and ChATfl/fl;Mb1-Cre mice (n = 5 for ChATfl/fl and n = 6 for Cre) respectively.

C-D - Body weight (C) and IWAT weight (D) of ChATfl/fl;LysM-Cre (n = 7 for ChATfl/fl, n = 8 for Cre in C, D), ChATfl/fl;Cd4-Cre (n = 6 for ChATfl/fl and n = 7 for Cre in C, D), ChATfl/fl;Mb1-Cre (n = 12 in C, n = 9 for ChATfl/fl, n = 8 for Cre in D) and littermate ChATfl/fl mice housed at RT.

E-G - mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl;LysM-Cre (n = 24 for ChATfl/fl_RT, n = 25 for Cre_RT, n = 20 for ChATfl/fl_CE, n = 20 for Cre_CE) (E), ChATfl/fl;Cd4-Cre (n = 17 for ChATfl/fl_RT, n = 22-23 for Cre_RT, n = 19 for ChATfl/fl_CE, n = 22 for Cre_CE) (F), ChATfl/fl;Mb1-Cre (n = 10 for ChATfl/fl_RT, n = 11 for Cre_RT, n = 9-12 for ChATfl/fl_CE, n = 13 for Cre_CE) (G) and littermate ChATfl/fl mice housed at RT or 4oC (CE) for 6 h. An insert graph in (E) highlights mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl and ChATfl/fl;LysM-Cre mice after 6 h CE.

H - LC/MS-MS was used to quantify Ach secretion from IWAT SVF cells isolated from ChATfl/fl and ChATfl/fl;LysM-Cre mice housed at RT or 4 h CE (n = 3). n.s.: not significant.

I - Immunoblotting for UCP1 and GAPDH (loading control) in IWAT from ChATfl/fl (n = 7) and ChATfl/fl;LysM-Cre (n = 5) mice after 6 h CE. BAT served as a positive control (PC) for UCP1 expression. Film was subjected to a short and long exposure and size (kDa) is marked on the right hand side. #: non-specific bands.

J - Basal and oligomycin-insensitive OCR of IWAT from cold-exposed ChATfl/fl (n = 11) and ChATfl/fl;LysM-Cre (n = 13) mice for 6 h.

K - Average whole-body oxygen consumption rate (OCR) of ChATfl/fl (n = 10) and ChATfl/fl;LysM-Cre (n = 14) mice housed in metabolic chambers at RT or CE for 6 h (from 9 a.m. to 3 p.m.).

L - Relative mRNA expression of Chrna2 and thermogenic genes in BAT of ChATfl/fl and ChATfl/fl;LysM-Cre mice housed at RT or 6 h CE (n = 12 for ChATfl/fl_RT, n = 13 for ChATfl/fl;LysM-Cre_RT, n = 15 for ChATfl/fl_CE, n = 14 for ChATfl/fl;LysM-Cre_CE).

Data information: For (B, E-G and L), mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. In (B-E, H and J-K), data are presented as mean ± SEM where *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed Student's t-test). In (E-G and L), data are presented as mean ± SEM and the letters "a", "b" and "c" indicate P < 0.05 between groups (one-way ANOVA).
Acetylcholine-synthesizing macrophages in subcutaneous fat are regulated by β2-adrenergic signaling (2021)
Figure 3

Figure 3. ChAMs link adrenergic signaling to beige fat activation.

A - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3).

B - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3).

C - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4).

D - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4).

E - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice treated with vehicle (veh) or 1 mg/kg NE for 2 h (n = 6).

F - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice treated with veh or 1 mg/kg NE for 2 h (n = 6).

G - Schematic describing the generation of ChAT-eGFP;Cx3cr1CreER-RFP mice by crossing ChAT-eGFP, Cx3cr1CreER, and Ai14 animals.

H - Left: Representative histograms showing RFP expression profile of liver monocyte-derived MΦ (MDMs) and Kupffer cells (KCs) in ChAT-eGFP;Cx3cr1CreER-RFP mice. Right: Ratio of liver KCs to MDMs that are labeled RFP- or RFP+ in ChAT-eGFP;Cx3cr1CreER-RFP mice (n = 3).

I - Left: Representative flow plots showing ChAT-eGFP+ and Cx3cr1CreER-RFP+ double positive MΦ (yellow gate) in the IWAT of ChAT-eGFP;Cx3cr1CreER-RFP mice. Right: Percentage of ChAT-eGFP+ MΦ that are RFP- or RFP+ in the IWAT of ChAT-eGFP;Cx3cr1CreER-RFP mice (n = 5).

J - Relative mRNA expression of Adrb1, Adrb2 and Adrb3 in IWAT MΦ sorted from WT mice (n = 4). Expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.

K - Relative mRNA expression of Adrb1, Adrb2 and Adrb3 in BMDMs (n = 3). mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.

L - Relative mRNA expression of Chat in BMDMs treated for 2 h with veh or 100 μM NE (n = 6). mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.

M - Total number of ChAT-eGFP+ cells in BMDMs derived from ChAT-eGFP and β-less ChAT-eGFP mice (n = 4). BMDMs were treated for 2 h with veh or 100 μM NE then an equal number of events (50,000) were analyzed by flow cytometry. n.s.: not significant.

Data information: In (A-F, H-I and L-M), data are presented as mean ± SEM where *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed Student's t-test). In (J-K), data are presented as mean ± SEM and the letters "a", "b" and "c" indicate P < 0.05 between groups (one-way ANOVA).
FAM111A Induces Nuclear Dysfunction in Disease and Viral Restriction (2020)
Figure 1

Figure 1. FAM111A localization in multiple cell lines.

A. Schematic representation of human FAM111A domains and mutations that compromise FAM111A functions. The FAM111A N-terminus contains a functional PCNA interaction motif (PIP box; Alabert et al., 2014()). The FAM111A C-terminal trypsin-like protease domain contains a typical H385-D439-S541 catalytic triad Unger et al., 2013(), and binds the C-terminus of the SV40 Large T antigen Fine et al., 2012Tarnita et al., 2019(, ). We used an S541A mutation to disrupt the catalytic triad and inactivate FAM111A's trypsin-like protease activity. Finally, the KCS2 and OCS patient mutations are indicated in red boxes. See text and Unger et al., 2013() for phenotypic details.

B. Confocal microscopy images of HeLa, RPE, and U2OS cell lines that were pre-extracted with Triton X-100 before fixed with formaldehyde and co-stained with DAPI and antibodies against PCNA and FAM111A. Scale bars: 10 μm

C. U2OS cells were stained with antibodies against PML and FAM111A and imaged by confocal microscopy. Scale bars: 10 μm.
FAM111A Induces Nuclear Dysfunction in Disease and Viral Restriction (2020)
Figure 2

Figure 2. The effect of overexpressing wild-type or mutant FAM111A on cell growth and cell cycle profiles in multiple cell lines.

A, C. HEK293, RPE, or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline to express wild-type or mutant FAM111A for 24 h (HEK293), 48 h (RPE), or 72h (U2OS). The colonies were fixed then stained with crystal violet. Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3). *p<0.05, **p<0.01, ****p<0.0001 (two-tailed unpaired t-test).

B, D. Cell-cycle profiles of HEK293, RPE and U2OS FAM111A-expressing cell lines that were induced with doxycycline for 48 h and analyzed by flow cytometry.
FAM111A Induces Nuclear Dysfunction in Disease and Viral Restriction (2020)
Figure 3

Figure 3. Caspase inhibitor Z-VAD-FMK inhibits PARP cleavage, but not cell death, in cells overexpressing FAM111AR569H.

A. HEK293 or U2OS/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from 105 cells was resolved by SDS-PAGE, and probed in western blots for both endogenous FAM111A (71 kDa) and MycBioID-FAM111A (110 kDa) as well as full length and cleaved PARP (116 and 89 kDa, respectively). The Tubulin western blot is a sample loading control.

B. HEK293/MycBioID-FAM111A cell lines with no treatment (- Dox), or treated with doxycycline, along with DMSO or 100 μM Z-VAD-FMK for 24 h (total inhibition of PARP cleavage was also observed when 50 μM Z-VAD-FMK was used). Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots with Myc, PARP, or Tubulin antibodies.

C. HEK293 cell lines were treated with doxycycline and Z-VAD-FMK for 24 h. The colonies were fixed then stained with crystal violet. Cell growth was quantitated as total area on ImageJ. Values are mean ± s.e.m. of independent experiments (n = 3).
FAM111A Induces Nuclear Dysfunction in Disease and Viral Restriction (2020)
Figure 4

Figure 4. NPC components are potential targets of FAM111A activity.

A. A subset of FAM111A interacting proteins identified by BioID screen is listed along with the number of peptide spectral counts observed for each protein.

B. A STRING diagram showing the interaction of NPC components identified in our FAM111A BioID screen.

C. Cells were treated with doxycycline along with DMSO or Z-VAD-FMK for 24 h. They were fixed with formaldehyde, and stained with NPC antibody Mab414 and DAPI. The images were captured by confocal microscopy. Scale bar: 10 μm.

D. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from an equal number of cells was incubated with magnetic streptavidin beads, and pull downs were analyzed by western blotting using GANP and Myc antibodies.

E. HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h, while growing in the presence or absence of 50 mM supplemental biotin. Total protein from whole cell lysates was incubated with magnetic streptavidin beads. Pulldowns were analyzed by western blotting using GANP and Myc antibodies.

F. DMSO or Z-VAD-FMK treated HEK293/MycBioID-FAM111A cell lines were induced with doxycycline for 24 h. Total protein from ~ 105 cells was resolved by SDS-PAGE, and probed in western blots using antibodies against NPC (Mab414), PARP, cleaved Caspase 3, Myc, or Tubulin.