EMBO/soda-nel huggingface dataset:
Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 (2022)
Figure 4

Figure 4. Sigmar1 interacts with SERCA2 and mediates its degradation.

(A) SERCA2 peptides identified through mass spectrometry are shown.

(B) HEK-293T cells were transfected with the indicated plasmids and then subjected to immunoprecipitation followed by western blotting to detect exogenous interactions between Sigmar1 and SERCA2.

(C) The lysates from mouse BMMs were incubated with either anti-Sigmar1 antibody or normal rabbit IgG, and the pellets were detected with the indicated antibodies.

(D) Western blots showing SERCA2 expression in HEK-293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg SERCA2 plasmid and Sigmar1 plasmid (0.125 μg, 0.25 μg, 0.5 μg, 1 μg).

(E) Upper: schematic representation of various Sigmar1 truncations. Bottom: mapping of Sigmar1 domains critical for SERCA2 binding. HEK-293T cells were transfected with different Sigmar1 truncations, and cell lysates were immunoprecipitated and subjected to western blotting.

(F) Upper: schematic representation of various SERCA2 truncations. Bottom: mapping of SERCA2 domains crucial for Sigmar1 binding. Different truncated SERCA2 plasmids were transfected into HEK-293T cells. After immunoprecipitation, the interaction between truncated SERCA2 and Sigmar1 was detected by western blotting. Red asterisks indicate specific SERCA2 truncation bands.

(G) Binding mode of SERCA2 (positions 314-807) on the SIGMAR1 homotrimer predicted by docking. Upper: overall structure of SERCA2 bound to SIGMAR1 in cartoon view. SERCA2 and SIGMAR1 are colored in wheat and light blue, respectively, and the chain identifiers of the SIGMAR1 homotrimer are labeled. Middle and bottom: detailed interaction network between SERCA2 and SIGMAR1. Key residues of SERCA2 (deep teal) and SIGMAR1 (pink) are displayed as sticks, and chain identifiers of residues are shown. H-bonds are displayed in red dashed lines, and the distances (acceptor to donor heavy atom) of H-bonds are labeled.

(H) Western blots showing Q615A mutant truncated SERCA2 expression in HEK-293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg Q615A mutant of truncated SERCA2 plasmid and Sigmar1 plasmid (0.125 μg, 0.25 μg, 0.5 μg, 1 μg).

(I) Interactions between Sigmar1 and WT or Q615 mutants of truncated SERCA2 were detected by Co-IP assays.

(J) TRAP staining to detect osteoclastogenesis of BMMs from WT and gKO mice treated with different siRNAs. Scale bars, 200 μm.

Data information: All results are representative data generated from at least three independent experiments.
Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 (2022)
Figure 5

Figure 5. Sigmar1 mediates SERCA2 degradation through the Hrd1/Sel1L-dependent ERAD pathway.

(A and B) Western blots showing SERCA2 expression in HEK-293T cells with or without Sigmar1 cotransfection treated with eeyarestatin I or MG132 at the indicated concentration. All inhibitors were applied to cells 8 hours prior to protein collection.

(C) Western blots showing changes in SERCA2 expression in HEK-293T cells in the presence of different doses of flag-tagged Hrd1 plasmids.

(D) Western blots showing alterations of SERCA2 expression in HEK-293T cells pretreated with negative control siRNA (si-NC) or Sel1L siRNA in the presence or absence of Sigmar1.

(E) Truncated myc-tagged SERCA2 ubiquitination levels in HEK-293T cells transfected with empty vector or Sigmar1 were analyzed by immunoprecipitation. Cells were treated with MG132 (10 μM) 8 hours before harvest.

(F) Expression of different truncated SERCA2 lysine mutants in HEK-293T cells transfected with different amounts of Sigmar1 plasmid.

(G) Expression of WT or 2KR (K460A and K541A) mutants of full-length SERCA2 in HEK-293T cells transfected with different amounts of Sigmar1 plasmid.

Data information: All results are representative data generated from at least three independent experiments.
Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 (2022)
Figure 6

Figure 6. Dimemorfan protects mice from established bone loss in various pathological models.

(A) Schematic illustrating the protocol for LPS-induced osteolysis and dimemorfan treatment.

(B) Micro-CT images of calvaria from mice that received sham or LPS injection with PBS or dimemorfan treatment. Scale bars, 1 mm.

(C) Quantification of bone volume/tissue volume (BV/TV) of calvaria from different groups (n = 6 biological replicates).

(D) H&E and TRAP staining of calvaria from the three groups. Scale bars, 100 μm.

(E and F) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates).

(G) The schematic illustrates the protocol for OVX-induced bone loss and dimemorfan treatment.

(H) Micro-CT images of proximal femurs from sham or ovariectomized mice with different treatments. Scale bars, 1 mm.

(I) Quantification of bone volume/tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 6 biological replicates).

(J) TRAP staining of femurs from the three groups. Scale bars, 200 μm.

(K and L) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates).

(M to O) Serum IL-1β, IL-6 and TNF-α concentrations measured by ELISA in the three groups (n = 5 biological replicates).

Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C, E-F, I, K-O) was used for statistical analysis.
Sigma-1 receptor attenuates osteoclastogenesis by promoting ER-associated degradation of SERCA2 (2022)
Figure 7

Figure 7. Dimemorfan inhibits osteoclast differentiation in hPBMCs.

(A) TRAP staining of hPBMCs treated with human RANKL (50 ng/mL) or dimemorfan for 12 days. Scale bars, 500 μm.

(B) Western blot analysis of NFATc1 expression during a 7-day induction of hPBMCs to osteoclasts.

(C) hPBMCs were induced to differentiate into osteoclasts for 7 days, and the relative mRNA levels of marker genes were evaluated by RT-qPCR (n=6 biological replicates).

Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C) was used for statistical analysis.
Targeting prominin2 transcription to overcome ferroptosis resistance in cancer (2021)
Figure 1

Figure 1. RSL3 and the lipid peroxidation metabolite 4HNE trigger an increase in PROM2 expression.

A) MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

B) MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either DMSO, 5uM RSL3 or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

C) MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

D) MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either EtOH, 25uM 4HNE or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. n=3 experiments per group. Exact p‑values are reported in the Appendix Table S1.

E) MCF10A cells were treated with either EtOH, 25uM 4HNE or 5uM RSL3 for 60 minutes. Isolated protein was assessed by immunoblotting for 4HNE, prominin2, and β-actin expression. Shown is one replicate of three independent experiments (n=3).

F) MCF10A cells were treated with DMSO or 100uM MDA for 60 and 120 minutes. Isolated protein was assessed by immunoblotting for prominin2, and β-actin expression. Shown is one replicate of three independent experiments (n=3).
Targeting prominin2 transcription to overcome ferroptosis resistance in cancer (2021)
Figure 2

Figure 2 4HNE activates p38 to upregulate prominin2.

A) MCF10A cells were treated with either DMSO, 25uM 4HNE or 5uM RSL3 for 60 minutes. Isolated protein was assessed by immunoblotting for phospho-p38 (Thr180/Tyr182) and total p38 expression. Shown is one replicate of three independent experiments (n=3).

B) MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes. Isolated protein was assessed by immunoprecipitating phospho-p38 (Thr180/Tyr182) and immunoblotting for 4HNE and phospho-p38 (Thr180/Tyr182). Shown is one replicate of three independent experiments (n=3).

C) MCF10A cells were pretreated for 15 minutes with either DMSO or 10uM BIRB, followed by 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

D) MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-p38 (Thr180/Tyr182) and total p38. Shown is one replicate of three independent experiments (n=3).

E) MCF10A cells were treated with RSL3 with either DMSO or 10uM BIRB. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.
Targeting prominin2 transcription to overcome ferroptosis resistance in cancer (2021)
Figure 3

Figure 3 4HNE activates HSF1 to induce prominin2 expression

A) HSF1 was identified as a strong occupier of the PROM2 promoter

B) MCF10A cells were treated with either 25uM 4HNE or 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and HSF1 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

C) MCF10A cells were pretreated for 15 minutes with either DMSO or 10uM BIRB, followed by 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

D) MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (S326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38 and β-actin expression. Shown is one replicate of three independent experiments (n=3).

E) MCF10A cells were plated on slides pre-coated with laminin. Cells were then pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Cells were stained for total HSF1 and counterstained with DAPI. Images were taken at 20x magnification. Scale bar is 50 microns. Shown is one replicate of three independent experiments (n=3).

F) HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO, EtOH, DMSO, 25uM 4HNE or 5uM RSL3. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).

G) HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO or RSL3 at the range of concentrations shown. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.
Targeting prominin2 transcription to overcome ferroptosis resistance in cancer (2021)
Figure 4

Figure 4 HSF1 inhibition prevents prominin2-mediated evasion of ferroptosis

A) MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

B) MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

C) MCF10A cells were plated on slides pre-coated with laminin. Cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB1 or RSL3 and KRIBB11. Cells were stained for total HSF1 and counterstained with DAPI. Images were taken at 20x magnification. Scale bar is 50 microns. Shown is one replicate of three independent experiments (n=3).

D) MCF10A cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. Isolated protein was assessed by immunoblotting for prominin2, phospho‑HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).

E) MCF10A cells were treated with either RSL3, FIN56 or IKE at the range of concentrations shown with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

F) HMLE cells were treated with IKE at the range of concentrations shown with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

G) S1 cells were treated with IKE at the range of concentrations shown with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.
Targeting prominin2 transcription to overcome ferroptosis resistance in cancer (2021)
Figure 6

Figure 6. Inhibition of HSF1 increases sensitivity to IKE in vivo.

A) Schematic of experimental design. Figure partially created using BioRender.

B) Tumor volume was measured daily by electronic caliper. Data are plotted as the mean ± SD for n=8 individual mice per group. P‑values were obtained by two‑way ANOVA with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.

C) Mouse weight was monitored daily and indicated no significant weight loss with IKE + KRIBB11. Data are plotted as the mean ± SD for n=8 individual mice per group.

D) Twenty-four hours after the last treatment, tumors were excised, frozen and sectioned. Each tumor was stained with Abs specific for either 4HNE, prominin2 or HSF1. Images were taken at 20x magnification. Higher‑magnification images (1.5x) are shown to the right of its corresponding inset (white box). Scale bar is 50 microns. Shown is one representative image for each condition.
RAD54 promotes Alternative Lengthening of Telomeres by mediating branch migration (2020)
Figure 1

Figure 1: RAD54 localizes to ALT telomeres in response to DNA damage

A Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines. White arrows indicate RAD54 foci that colocalize with telomeres. Scale bars = 10 µm

B Quantification of data in A. A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere. At least 100 cells were counted per cell line per repeat. For SaOS2, NOS, SJSA1, HeLa LT n = 3. For Cal72, U2OS, HuO9 n = 4. Values shown are mean±sd

C Combined IF and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µm

D Quantification of HuO9 data from C. RAD54 foci were detected using particle analysis in Fiji. Total RAD54 foci were counted. RAD54 foci were considered in APBs if they colocalized with both PML and telomere. At least 100 cells counted per repeat, n = 3. Values shown are mean±sd

E Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs

F Combined IF and DNA FISH for RAD54 (IF) and telomeres (FISH) in U2OS-TRF1-FOK1-WT or U2OS-TRF1-FOK1-D450A (nuclease dead) cells. Cells were treated as in E immediately prior to staining. Scale bars = 10 µm

G Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. At least a total of 300 cells were counted from 3 repeated experiments. Values shown are mean±sem. Values were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test.

H Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres. Values shown are mean±sem of n=3 biological replicates. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.
RAD54 promotes Alternative Lengthening of Telomeres by mediating branch migration (2020)
Figure 2

Figure 2: RAD54 is dispensable for synapsis at ALT telomeres

A Combined IF and DNA FISH for RAD51 (IF) and telomeres (FISH) in SaOS2 or HuO9 cells. Cells were transfected with 20nM siRAD54#2 for 48 hours immediately prior to staining. Scale bars = 10 µm

B Quantification of A. A cell was counted as positive it is contained at least 1 colocalization event between RAD51 and telomeres. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.

C ChIP for telomeric DNA associated with RAD51. DNA dot blot probed with DIG labeled telomere probe or Alu probe. HuO9 cells were transfected with 20nM siRAD54 #2, or 15µg myc-TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody (Mock, siRAD54 conditions) or myc antibody (myc-TRF2 positive control).

D Quantification of (CCCTAA)4 dot blot performed in C. IP values were normalized to loading (IP/input) and then normalized to mock control. Values shown are mean±sd of n=3 biological replicates. Values were compared using two-tailed unpaired student's t-test.

E Western blot of samples used for ChIP in C-D.

F Combined IF and DNA FISH for GFP (IF) and telomeres (FISH) in SaOS2 and HuO9 cells. Cells were forward transfected with Polη-GFP, and then after 24 hours transfected with 20nM siRAD54#2. Cells were stained for GFP and telomeres 48 hours after siRNA transfection. Scale bars = 10 µm

G Quantification of data in F. GFP negative cells were excluded from analysis. Cells were considered positive if they showed 1 or more colocalization events between GFP (Polη) and telomeres. At least 50 GFP positive cells were counted per condition per repeat, n=3. Values shown are mean±sd. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.