Journal: BMC cancer

Article Title: Identification of genes inducing resistance to ionizing radiation in human rectal cancer cell lines: re-sensitization of radio-resistant rectal cancer cells through down regulating NDRG1.

doi: 10.1186/s12885-018-4514-3

Figure Lengend Snippet: Fig. 3 Potential target genes for acquiring radio-resistance were further sorted with FC > 3 threshold, and five genes (NDRG1, ERRFI1, H19, MPZL3, and UCA1) were selected. RT-PCR (a) and real-time PCR (b, c, d, e, and f) confirmed that the mRNA expression patterns of the five genes were analogous to the microarray analysis (P: parental rectal cancer cell line, R: radio-resistant rectal cancer cell line, N: negative control)

Article Snippet: Primary antibodies against NDRG1 (abcam, Cambridge, United Kingdom) (1:5000), ERRFI1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:1000), PARP (BD Biosciences, San Jose, CA, USA) (1:1000), Caspase-3 (abcam, Cambridge, United Kingdom) (1:2000), and β-actin (Applied Biological Materials Inc., Richmond, BC, Canada) (1:5000) were diluted with 1.5% skim milk (BD Biosciences, CA, USA) containing 0.5% Tween 20 and introduced to the membrane.

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Microarray, Negative Control

Journal: BMC cancer

Article Title: Identification of genes inducing resistance to ionizing radiation in human rectal cancer cell lines: re-sensitization of radio-resistant rectal cancer cells through down regulating NDRG1.

doi: 10.1186/s12885-018-4514-3

Figure Lengend Snippet: Fig. 4 Subcellular localization and expression of the ERRFI1 and NDRG1 proteins in SNU-503 and SNU-503R80Gy

Article Snippet: Primary antibodies against NDRG1 (abcam, Cambridge, United Kingdom) (1:5000), ERRFI1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:1000), PARP (BD Biosciences, San Jose, CA, USA) (1:1000), Caspase-3 (abcam, Cambridge, United Kingdom) (1:2000), and β-actin (Applied Biological Materials Inc., Richmond, BC, Canada) (1:5000) were diluted with 1.5% skim milk (BD Biosciences, CA, USA) containing 0.5% Tween 20 and introduced to the membrane.

Techniques: Expressing

Journal: BMC cancer

Article Title: Identification of genes inducing resistance to ionizing radiation in human rectal cancer cell lines: re-sensitization of radio-resistant rectal cancer cells through down regulating NDRG1.

doi: 10.1186/s12885-018-4514-3

Figure Lengend Snippet: Fig. 5 Expression level of the ERRFI1 and its role in cell proliferation under irradiation in the SNU-503 and SNU-503R80GY cell lines. a The protein expression of ERRFI1 in three pairs of the induced rectal cancer cell lines was confirmed by Western Blot analysis (P: Parental rectal cancer cell line, R: Radio-resistant rectal cancer cell line) were performed. b The knock-down efficiency of siERRFI1 in SNU-503R80Gy cell line was accessed by RT-PCR. Cell counting c and MTT assay d were performed 96 h after siERRFI1 transfection (N: Negative control)

Article Snippet: Primary antibodies against NDRG1 (abcam, Cambridge, United Kingdom) (1:5000), ERRFI1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:1000), PARP (BD Biosciences, San Jose, CA, USA) (1:1000), Caspase-3 (abcam, Cambridge, United Kingdom) (1:2000), and β-actin (Applied Biological Materials Inc., Richmond, BC, Canada) (1:5000) were diluted with 1.5% skim milk (BD Biosciences, CA, USA) containing 0.5% Tween 20 and introduced to the membrane.

Techniques: Expressing, Irradiation, Western Blot, Knockdown, Reverse Transcription Polymerase Chain Reaction, Cell Counting, MTT Assay, Transfection, Negative Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 1 ERRFI1 deficiency ameliorated IR-induced hepatic injury and hepatocellular apoptosis. (A) Schematic diagram showed the hepatocyte-specific ERRFI1 knockout strategy and ischemia reperfusion model. (B, C) Western blot analysis of ERRFI1 protein level in liver tissues from wild type (WT) mice and hepatocyte-specific ERRFI1-knockout (ERRFI1-HKO) mice with sham treatment or ischemia for 90 min followed by reperfusion for 6 h, and quantita tive analysis is shown. (D) Liver function assessed by ALT and AST of mice with different treatment. (E) Liver pathology was determined by H&E staining (scale bar: 100 μm). (F) Suzike’s injury score was used to assess the degree of injury based on H&E staining. (G, H) TUNEL staining of apoptotic cells in liver tissues from WT mice and ERRFI1-HKO mice under different conditions (scale bar: 50 μm), and quantification showing the percentage of apoptotic cells. (I, J) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in liver tissues from WT mice and ERRFI1-HKO mice after IR injury. For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 2 ERRFI1 deficiency protected against hepatic IR-induced ferroptosis. (A) Intracellular ROS level was determined by DCFH-DA staining after hepatic IR. (B) Immunohistochemical staining of 8-OHdG in liver tissues (scale bar: 50 μm). (C, D) The content of MDA and the level of GSH in the livers of mice subjected to sham treatment or to an induction of IR. (E) Hepatic Fe2+ content in each group. (F) The mRNA levels of ACSL4, SLC7A11, and GPX4 in liver tissues of mice with different treatments. (G) Representative immunohistochemical images of ACSL4, SLC7A11, and GPX4 in liver tissues (scale bar: 50 μm). For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Staining, Immunohistochemical staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 3 Knockdown of ERRFI1 inhibited apoptosis of hepatocytes induced by hypoxic-reoxygenation. (A) Expression of ERRFI1 in L-02 cells following hy poxia/reoxygenation (H/R) and ERRFI1 knockdown at the mRNA level was determined by real-time PCR. (B) Cell viability of ERRFI1-silenced L-02 cells after OGD/R exposure. (C, D) Apoptosis-positive cells were detected by TUNEL staining (scale bar: 50 μm). (E, F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in L-02 cells under indicated conditions. For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 4 Knockdown of ERRFI1 suppressed OGD/R-induced ferroptosis in hepatocytes. (A) ROS level in ERRFI1-silenced L-02 cells exposed to hypoxia/ reoxygenation. (B) Flow cytometry analysis of lipid peroxidation using C11-BODIPY 581/591 in L-02 cells under indicated conditions. (C, D) MDA content and GSH level in L-02 cells cultured under indicated conditions. (E) Fe2+ content in ERRFI1-silenced L-02 cells after OGD/R exposure was determined. (F) Real-time PCR showed the mRNA levels of ACSL4, SLC7A11, and GPX4 in response to ERRFI1 knockdown under H/R conditions. (G, H) Fluorescence im munostaining of ACSL4, SLC7A11, and GPX4 in L-02 cells transfected with sh-ERRFI1 during H/R injury (scale bar: 20 μm). For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Knockdown, Flow Cytometry, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence, Transfection

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 5 ERRFI1 directly interacted with GRB2 and maintained its stability by hindering its proteasomal degradation. (A-C) Transcript and protein levels of GRB2 in ERRFI1-silenced L-02 cells were detected. (D) Co-immunoprecipitation of ERRFI1 and GRB2. L-02 cells were subjected to GRB2 immunoprecipita tion and subsequent immunoblotting of ERRFI1 and GRB2. (E, F) L-02 cells were transfected with sh-ERRFI1 and treated with CHX for the indicated times. Western blot analysis showed the expression of GRB2. (G, H) The expression of GRB2 in sh-ERRFI1-transfected L-02 cells with or without MG132 treatment. For statistical analysis, Student’s t test was used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 6 ERRFI1 facilitated OGD/R-induced injury of hepatocytes in a GRB2-dependent manner. (A-D) L-02 cells were transfected with GRB2 overexpression plasmid or vector plasmid, followed by 4 h of hypoxia and 12 h of reoxygenation. Cell apoptosis was detected by TUNEL staining. Scale bar: 20 μm (A, B). Lipid peroxidation was measured using C11-BODIPY 581/591 (C). Fe2+ content was determined (D). (E, F) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 20 µM Z-VAD-FMK (ZVF, an apoptosis inhibitor). L-02 cells were treated with 5 µM camptothecin (CPT, an apoptosis inducer) as positive control at the same time as OGD/R stimulation. TUNEL staining of L-02 cells under indicated conditions was performed (scale bar: 20 μm). (G-L) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 5 µM ferrostatin-1 (Fer-1, a ferroptotic inhibitor). L-02 cells were treated with 10 µM erastin (a ferroptotic inducer) as positive control at the same time as OGD/R stimulation. DCFH-DA staining was used to detect ROS production in cells (G). Fe2+ content in cells was measured by a commercial kit (H). The protein expression of GPX4 in cells was analyzed by immunofluorescence. Scale bar: 20 μm (I, K). Cell death was detected by propidium iodide (PI) staining. Scale bar: 50 μm (J, L). For statistical analysis, student’s t test and one-way ANOVA were used (n = 3)

Article Snippet: The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

Techniques: Transfection, Over Expression, Plasmid Preparation, TUNEL Assay, Staining, Positive Control, Expressing, Immunofluorescence

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 1 ERRFI1 deficiency ameliorated IR-induced hepatic injury and hepatocellular apoptosis. (A) Schematic diagram showed the hepatocyte-specific ERRFI1 knockout strategy and ischemia reperfusion model. (B, C) Western blot analysis of ERRFI1 protein level in liver tissues from wild type (WT) mice and hepatocyte-specific ERRFI1-knockout (ERRFI1-HKO) mice with sham treatment or ischemia for 90 min followed by reperfusion for 6 h, and quantita tive analysis is shown. (D) Liver function assessed by ALT and AST of mice with different treatment. (E) Liver pathology was determined by H&E staining (scale bar: 100 μm). (F) Suzike’s injury score was used to assess the degree of injury based on H&E staining. (G, H) TUNEL staining of apoptotic cells in liver tissues from WT mice and ERRFI1-HKO mice under different conditions (scale bar: 50 μm), and quantification showing the percentage of apoptotic cells. (I, J) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in liver tissues from WT mice and ERRFI1-HKO mice after IR injury. For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knock-Out, Western Blot, Staining, TUNEL Assay

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 2 ERRFI1 deficiency protected against hepatic IR-induced ferroptosis. (A) Intracellular ROS level was determined by DCFH-DA staining after hepatic IR. (B) Immunohistochemical staining of 8-OHdG in liver tissues (scale bar: 50 μm). (C, D) The content of MDA and the level of GSH in the livers of mice subjected to sham treatment or to an induction of IR. (E) Hepatic Fe2+ content in each group. (F) The mRNA levels of ACSL4, SLC7A11, and GPX4 in liver tissues of mice with different treatments. (G) Representative immunohistochemical images of ACSL4, SLC7A11, and GPX4 in liver tissues (scale bar: 50 μm). For statistical analysis, one-way ANOVA was used (n = 6)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Staining, Immunohistochemical staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 3 Knockdown of ERRFI1 inhibited apoptosis of hepatocytes induced by hypoxic-reoxygenation. (A) Expression of ERRFI1 in L-02 cells following hy poxia/reoxygenation (H/R) and ERRFI1 knockdown at the mRNA level was determined by real-time PCR. (B) Cell viability of ERRFI1-silenced L-02 cells after OGD/R exposure. (C, D) Apoptosis-positive cells were detected by TUNEL staining (scale bar: 50 μm). (E, F) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in L-02 cells under indicated conditions. For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, TUNEL Assay, Staining, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 4 Knockdown of ERRFI1 suppressed OGD/R-induced ferroptosis in hepatocytes. (A) ROS level in ERRFI1-silenced L-02 cells exposed to hypoxia/ reoxygenation. (B) Flow cytometry analysis of lipid peroxidation using C11-BODIPY 581/591 in L-02 cells under indicated conditions. (C, D) MDA content and GSH level in L-02 cells cultured under indicated conditions. (E) Fe2+ content in ERRFI1-silenced L-02 cells after OGD/R exposure was determined. (F) Real-time PCR showed the mRNA levels of ACSL4, SLC7A11, and GPX4 in response to ERRFI1 knockdown under H/R conditions. (G, H) Fluorescence im munostaining of ACSL4, SLC7A11, and GPX4 in L-02 cells transfected with sh-ERRFI1 during H/R injury (scale bar: 20 μm). For statistical analysis, one-way ANOVA was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Knockdown, Flow Cytometry, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence, Transfection

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 5 ERRFI1 directly interacted with GRB2 and maintained its stability by hindering its proteasomal degradation. (A-C) Transcript and protein levels of GRB2 in ERRFI1-silenced L-02 cells were detected. (D) Co-immunoprecipitation of ERRFI1 and GRB2. L-02 cells were subjected to GRB2 immunoprecipita tion and subsequent immunoblotting of ERRFI1 and GRB2. (E, F) L-02 cells were transfected with sh-ERRFI1 and treated with CHX for the indicated times. Western blot analysis showed the expression of GRB2. (G, H) The expression of GRB2 in sh-ERRFI1-transfected L-02 cells with or without MG132 treatment. For statistical analysis, Student’s t test was used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: ERRFI1 exacerbates hepatic ischemia reperfusion injury by promoting hepatocyte apoptosis and ferroptosis in a GRB2-dependent manner.

doi: 10.1186/s10020-024-00837-4

Figure Lengend Snippet: Fig. 6 ERRFI1 facilitated OGD/R-induced injury of hepatocytes in a GRB2-dependent manner. (A-D) L-02 cells were transfected with GRB2 overexpression plasmid or vector plasmid, followed by 4 h of hypoxia and 12 h of reoxygenation. Cell apoptosis was detected by TUNEL staining. Scale bar: 20 μm (A, B). Lipid peroxidation was measured using C11-BODIPY 581/591 (C). Fe2+ content was determined (D). (E, F) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 20 µM Z-VAD-FMK (ZVF, an apoptosis inhibitor). L-02 cells were treated with 5 µM camptothecin (CPT, an apoptosis inducer) as positive control at the same time as OGD/R stimulation. TUNEL staining of L-02 cells under indicated conditions was performed (scale bar: 20 μm). (G-L) L-02 cells cotransfected with sh-ERRFI1 and GRB2 overexpression plasmid were subjected to OGD/R stimulation in the presence or absence of 5 µM ferrostatin-1 (Fer-1, a ferroptotic inhibitor). L-02 cells were treated with 10 µM erastin (a ferroptotic inducer) as positive control at the same time as OGD/R stimulation. DCFH-DA staining was used to detect ROS production in cells (G). Fe2+ content in cells was measured by a commercial kit (H). The protein expression of GPX4 in cells was analyzed by immunofluorescence. Scale bar: 20 μm (I, K). Cell death was detected by propidium iodide (PI) staining. Scale bar: 50 μm (J, L). For statistical analysis, student’s t test and one-way ANOVA were used (n = 3)

Article Snippet: Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.).

Techniques: Transfection, Over Expression, Plasmid Preparation, TUNEL Assay, Staining, Positive Control, Expressing, Immunofluorescence

Journal: iScience

Article Title: TRPA5 encodes a thermosensitive ankyrin ion channel receptor in a triatomine insect

doi: 10.1016/j.isci.2024.109541

Figure Lengend Snippet: Thermodynamics of RpTRPA5B temperature-activated currents (A–C) Experimental workflow. (A) Each TRP channel subcloned in the pFRT-TO-FLAG-T2A-mRuby2 expression cassette , was transfected in HEK293T cells seeded at low density and incubated at 37°C for 48 h. Cells were then prepared for patch-clamp recording by seeding in a 30-mm 2 culture dish overlaid with round glass cover slips and incubated at 30°C. (B) Electrophysiology recordings took place after 24–48 h using an optical fiber-based setup adapted after Yao et al. 2010, designed to couple manual patch-clamp recordings with fiber optics as a way to provide controllable optical and thermal stimulations to individual cells expressing candidate thermosensitive receptor proteins. The setup consists of a fiber launch system combining a high-power optical fiber tuned to near-infrared wavelengths (λc = 1,460 nm (+/−20 nm), Po = 4.8 W), a visible alignment laser (red), and a laser diode controller, forming a PID control loop using the patch-clamp current as the feedback signal. (C) During the experiment, a laser spot is aligned with one single patched cell (see Figure S6 ) stably expressing the membrane receptor protein of interest in the coverslip placed in the recording chamber. (D) Upper panel , current traces through the open patch-clamp pipette in response to temperature calibration steps from room temperature up to 71°C elicited by increments in the IR laser voltage input (see ). Each 700 ms voltage pulse is represented in different colors for the different temperatures calculated from the open pipette currents. Lower panel , representative recording of non-transfected cells; these cells did not show robust temperature-elicited currents, like negative cells on the recording plate. (E) Whole-cell currents evoked by temperature steps from HEK293T cells expressing rat TRPV1 (heat-activated mammalian vanilloid thermoTRP); cells were held at −30 mV during the recording. (F) Whole-cell currents evoked by temperature steps from HEK293T cells expressing dTRPA1-D (holding potential of −30 mV). The sinusoidal pattern observed within the current curves is inherent to the cyclic modulation of the laser’s rapid “on-off” cycles. (G) Whole-cell currents evoked by temperature steps in HEK293T cells expressing RpTRPA5B; cells were held at −30 mV. (H) Current-temperature relationship for RpTRPA5B whole-cell current was normalized by cell membrane capacitance (current density); the red line corresponds to a modified Boltzmann function that includes the leak and unitary current temperature dependence (see ). (I) Fraction of RpTRPA5B channels in the open state (open probability, P o ) as a function of the temperature. The Po vs. 1/T was fitted to a Boltzman function with the midpoint of activation (T 0.5 ) reached at 58.6°C. (J) van’t Hoff plot estimates of RpTRPA5B with an activation enthalpy of the endothermic transition at 92 kcal/mol and an entropic change associated with the temperature activation process at 274 cal/mol∗K at −30 mV. (K) Coupling between enthalpic (ΔH) and entropic (ΔS) changes for each one of the experiments recorded. (L) Free energy (ΔG) associated with the activation process as a function of temperature for RpTRP5AB channels. The receptor activation is associated with small free energy changes, as reported before for other families of mammalian thermoTRP receptors. ΔG was calculated as -RT∗ln(Keq). Data are represented as mean ± standard error.

Article Snippet: To program fast pseudo-transient temperature changes, the patch pipette current was used to read the temperature changes in real-time as the feedback to the laser diode controller (LDC-37620, ILX Lightwave) to perform proportional-integral-derivative (PID) control of the driving current of the laser diode.

Techniques: Expressing, Transfection, Incubation, Patch Clamp, Control, Stable Transfection, Membrane, Transferring, Modification, Activation Assay