DOI: 10.5061/dryad.18931zd9c
title: Data from: Brown adipose tissue and skeletal muscle coordinately contribute to thermogenesis in mice

description: Endotherms increase the rate of metabolism in metabolic organs as one
 strategy to cope with a decline in the temperature of the external
 environment. However, an additional major contributor to maintenance of
 body temperature in a cold environment is contraction-based thermogenesis
 in skeletal muscle. Here, we show that impairment of hind limb muscle
 contraction by cast immobilization induced a loss of function of skeletal
 muscle and activated brown adipose tissue (BAT) thermogenesis as a
 compensatory mechanism. BAT utilizes free branched-chain amino acids
 (BCAAs) derived from skeletal muscle as an energy substrate for
 thermogenesis, and interleukin-6 released by skeletal muscle stimulates
 BCAA production in muscle for support of BAT thermogenesis. Additionally,
 this thermoregulatory system between BAT and skeletal muscle may also play
 an important role in response to cold temperatures or acute stress. Our
 findings suggest that BAT and skeletal muscle cooperate to maintain body
 temperature in endotherms.
description: # Data from: Brown adipose tissue and skeletal muscle coordinately
 contribute to thermogenesis in mice Dataset DOI:
 [10.5061/dryad.18931zd9c](https://doi.org/10.5061/dryad.18931zd9c) ##
 Description of the data and file structure This dataset was generated to
 investigate physiological compensatory mechanisms associated with skeletal
 muscle dysfunction induced by hindlimb immobilization in male C57BL/6J
 mice. We analyzed metabolic changes in skeletal muscle and brown adipose
 tissue (BAT) using CE-TOFMS–based metabolomics, real-time PCR for gene
 expression, and radiolabeled tracer experiments to examine amino acid
 dynamics. To explore the role of interleukin-6 (IL-6) as a regulatory
 factor in muscle–BAT crosstalk, similar analyses were performed in IL-6
 knockout mice. ### Files and variables **Figure_1_source_data_1.xlsx**
 **Description:**  **Figure 1:**  (A) Time course of rectal core body
 temperature of cast-immobilized (gypsum, n = 6) and control (n = 5) mice
 subjected to a cold (4°C) challenge test. The test was performed 7 days
 after bilateral immobilization of hind limbs. (B) Rectal core body
 temperature of cast-immobilized (n = 5) and control (n = 5) mice during
 cold (4°C) exposure at 24 h after bilateral cast immobilization. (C)
 Decline in rectal temperature of cast-immobilized and control (n = 9) mice
 after cold (4°C) exposure for 4 h performed 2 h after unilateral (UL, n
 =8) or bilateral (BL, n = 9) immobilization. (D) Reverse transcription
 (RT) and real-time polymerase chain reaction (PCR) analysis of the
 *Ucp1/Gapdh* mRNA abundance ratio in iBAT of control mice and mice
 subjected to bilateral cast immobilization for the indicated times (n = 7
 to 13 per group). (F–G) Immunoblot analysis of PGC-1a (n = 3 or 4 per
 group) and UCP1 (n = 6 per group) in iBAT of control mice and mice
 subjected to bilateral cast immobilization for the indicated times. GAPDH
 was examined as a loading control. Representative blots (E) and
 densitometric quantitation of the PGC-1a/GAPDH (F) and UCP1/GAPDH (G) band
 intensity ratios are shown. (H) Noradrenaline concentrations in iBAT of
 cast-immobilized (n = 4) and control (n = 4) mice after subsequent biteral
 cast immobilization. (I) RT and real-time PCR analysis of the *Ucp1/Gapdh*
 mRNA abundance ratio in iBAT subjected to surgical denervation or sham
 surgery, either in control mice or in mice at 24 h after subsequent
 bilateral cast immobilization (n = 6–9 per group). (J) Rectal core body
 temperature of mice as in (I) subjected to cold (4°C) exposure for 1 h (n
 = 3 or 4 per group). **Figure 1_figure supplement 1:** (A–C) Weight of
 individual soleus (A), gastrocnemius (B), and extensor digitorum longus
 (EDL) muscles of control mice and mice subjected to bilateral cast
 immobilization of hind limbs for the indicated times (n = 5-7 per group).
 (D and E) Locomotor activity during light (D) and dark (E) phases for
 control mice and mice subjected to bilateral cast immobilization for the
 indicated times (n = 6 per group). (F) Rectal core body temperature of
 cast-immobilized (n = 6) and control (n = 4) mice at room temperature
 after bilateral cast immobilization. (G) RT and real-time PCR analysis of
 the expression of UCP1 genes in iBAT of control and cast-immobilized mice
 subjected (or not) to cold exposure at 4°C for 4 h (n = 4-9 per group).
 (H-K) RT and real-time PCR analysis of *Ucp2*, *Ucp3*, *Sln* and *Ppagc1a*
 expression in iBAT of control and cast-immobilized mice subjected (or not)
 to cold exposure at 4°C for 4 h (n = 4-9 per group). (L) Locomotor
 activity during cold (4°C) exposure for 4 h determined for control mice
 and mice subjected to unilateral (UL) or bilateral (BL) cast
 immobilization (n = 5 per group). These data were obtained in conjunction
 with those in Figure1C. **Figure 1_figure supplement 2:** (A)
 Concentrations of carbohydrate metabolites (acetyl CoA divalent, succinic
 acid, fumaric acid and malic acid) in soleus and EDL of control mice and
 mice subjected to bilateral cast immobilization for the indicated times.
 Results are presented as heat maps of the log~10~ value of fold change for
 cast-immobilized mice relative to control mice and are are shown
 individually for three mice of each group. (B and C) RT and real-time PCR
 analysis of *Ucp3* and *Ucp2* expression, respectively, in soleus of
 control mice and mice subjected to bilateral cast immobilization for the
 indicated times (n = 5-6 per group). (D and E) RT and real-time PCR
 analysis of *Sln* and *Camk2a* expression, respectively, in soleus of
 control mice and mice subjected to bilateral cast immobilization for the
 indicated times (n = 6 per group). (F and G) RT and real-time PCR analysis
 of *Ppargc1a*, and *Tfam* expression, respectively, in soleus of control
 mice and mice subjected to bilateral cast immobilization for the indicated
 times (n = 6 per group). (I) Food intake of control mice and mice
 subjected to bilateral cast immobilization for the indicated times (n =
 6–9 per group). **Figure_2_source_data_1.xlsx** **Description:**  **Figure
 2:** (A and B) Concentrations of carbohydrate metabolites (A) and amino
 acids (B) in sham-operated or denervated iBAT of mice without (control) or
 at the indicated times after bilateral cast immobilization. The
 metabolomics data are presented as heat maps corresponding to the log~10~
 value of fold change in cast-immobilized mice relative to the
 corresponding control mice and are means of four or five mice in each
 group. TCA, tricarboxylic acid cycle. (C and D) RT and real-time PCR
 analysis of mRNA abundance for the fatty acid transporter CD36 and the
 succinate transporter SLC25A10 in sham-operated or denervated iBAT of
 control mice or mice subjected to bilateral cast immobilization for 24 h
 (n = 6 per group). (E) Succinate content of sham-operated or denervated
 iBAT of control mice or mice subjected to bilateral cast immobilization
 for 24 h (n = 3–5 per group). (F and G) Oxygen consumption rate (VO~2~)
 and respiratory exchange ratio (RER), respectively, for control mice and
 mice subjected to bilateral cast immobilization for 7 days (n = 6 per
 group). (H) Weight of epididymal white adipose tissue (eWAT) for control
 mice and mice subjected to bilateral cast immobilization for the indicated
 times (n = 5–7 per group). (I) Hepatic glycogen content for control mice
 and mice subjected to bilateral cast immobilization for the indicated
 times (n = 6–10 per group). **Figure 2_figure supplement 1:** (A) Pathway
 analysis of metabolites upregulated in iBAT by cast immobilization for 10
 h. (B and C) Oxygen consumption rate (VO~2~) and RER, respectively, for
 mice with denervated iBAT subjected (or not) to bilateral cast
 immobilization for 7 days (n = 3 per group). Quantitative data other than
 in (A) are means ± SEM. *p &lt; 0.05, **p &lt; 0.01, as determined by
 two-way ANOVA followed by Tukey’s post hoc test or the post hoc
 paired/unpaired t test with Bonferroni’s correction (B and C). **Figure
 2_figure supplement 2:** (A and B) RT and real-time PCR analysis of G6Pase
 (*G6pc1*) and PEPCK (*Pck1*) gene expression in the liver of control mice
 and mice subjected to bilateral cast immobilization for the indicated
 times (n = 5 or 6 per group). (C) Concentrations of carbohydrate
 metabolites in the liver of control mice and mice subjected to bilateral
 cast immobilization for the indicated times. The metabolomics data are
 presented as heat maps corresponding to the log~10~ value of fold change
 in cast-immobilized mice relative to control mice. Results are means of
 three mice per group. (D) Serum concentration of noradrenaline in control
 mice and mice subjected to bilateral cast immobilization for the indicated
 times (n = 5–7 per group). (E) Serum concentration of corticosterone for
 control mice and mice subjected to bilateral cast immobilization for the
 indicated times (n = 6–8 per group). (F) RT and real-time PCR analysis of
 *Crh* expression in the hypothalamus of control mice and mice subjected to
 bilateral cast immobilization for the indicated times (n = 5–8 per group).
 **Figure_3_source_data_1.xlsx** **Description:**  **Figure 3:** (A and B)
 Metabolomics analysis of amino acid concentrations in soleus and EDL
 muscles (A) as well as in serum and the liver (B) of control mice and mice
 subjected to bilateral cast immobilization for the indicated times.
 Results are presented as heat maps of the log~10~ value of fold change for
 cast-immobilized mice relative to control mice and are means of three mice
 per group. (C) Organ-specific [^3^H]leucine uptake in control mice and
 mice subjected to bilateral cast immobilization for 24 h (n = 4–6 per
 group). sWAT, subcutaneous WAT. (D) RT and real-time PCR analysis of
 SLC25A44 gene expression in sham-operated or denervated iBAT of control
 mice and mice subjected to subsequent bilateral cast immobilization for 24
 h (n = 6 per group). **Figure 3_figure supplement 1.** (A and B) RT and
 real-time PCR analysis of *Fbxo32* (Atrogin-1) and *Trim63* (MuRF-1)
 expression in the soleus muscle of control mice and mice subjected to
 bilateral cast immobilization for the indicated times (n = 6 per group).
 (C–H) RT and real-time PCR analysis of SLC1A5, SLC7A5, SLC38A2, SLC43A1,
 BCAT2, and BCKHDA gene expression, respectively, in iBAT of control mice
 or mice subjected to bilateral cast immobilization for the indicated times
 (n = 3–6 per group). (I–M) RT and real-time PCR analysis of gene
 expression for SLC1A5, SLC7A5, SLC38A2, SLC43A1, and BCKHDA, respectively,
 in the liver of control mice or mice subjected to bilateral cast
 immobilization for the indicated times (n = 6 per group). (N–S) RT and
 real-time PCR analysis of gene expression for SLC1A5, SLC7A5, SLC38A2,
 SLC43A1, BCAT2, and BCKHDA in soleus of control mice or mice subjected to
 bilateral cast immobilization for the indicated times (n = 6 per group). 
 **Figure 3_figure supplement 2.** (A–C) RT and real-time PCR analysis of
 SLC25A44 gene expression in iBAT (A), liver (B), and soleus (C) of control
 mice or mice subjected to bilateral cast immobilization for the indicated
 times (n = 6 per group). (D–J) RT and real-time PCR analysis of gene
 expression for SLC25A44, SLC1A5, SLC7A5, SLC38A2, SLC43A1, BCAT2, and
 BCKDHA, respectively, in denervated iBAT of control mice or mice subjected
 to bilateral cast immobilization for the indicated times (n = 4–6 per
 group).  **Figure_4_source_data_1.xlsx** **Description:**  **Figure 4:**
 (A and B) RT and real-time PCR analysis of IL-1b, IL-6, IL-10, IL-15,
 TNF-a, MCP-1, BMP8B, FGF21, and irisin gene expression in iBAT (A) or
 soleus (B) of control mice or mice at 10 h (iBAT) or 24 h (soleus) after
 bilateral cast immobilization (n = 5 or 6 per group). (C and D) RT and
 real-time PCR analysis of *Il6* expression in iBAT (C) and soleus (D) of
 control mice and mice at the indicated times after bilateral cast
 immobilization (n = 3–7 per group). (E–L) RT and real-time PCR analysis of
 *Saa3* (E, G, I, and K) and *Socs3* (F, H, J, and L) mRNA abundance in
 iBAT (E and F), soleus (G and H), liver (I and J), and eWAT (K and L) of
 control mice and mice at the indicated times after bilateral cast
 immobilization (n = 3–7 per group) (M) RT and real-time PCR analysis of
 *Il6* expression, respectively, in denervated iBAT of control mice or mice
 at the indicated times after bilateral cast immobilization (n = 4-7 per
 group). (N) Serum IL-6 concentration in control mice or mice at the
 indicated times after bilateral cast immobilization (n = 6 per group).
 **Figure 4_figure supplement 1:** (A–G) RT and real-time PCR analysis of
 TNF-a, MCP-1, IL-1β, IL-10, IL-15, FGF21, and BMP8B gene expression,
 respectively, in iBAT of control mice and mice at the indicated times
 after bilateral cast immobilization (n = 3–6 per group). (H–N) RT and
 real-time PCR analysis of TNF-a, MCP-1, IL-1b, IL-10, IL-15, FGF21, and
 irisin gene expression, respectively, in soleus of control mice and mice
 at the indicated times after bilateral cast immobilization (n = 5 or 6 per
 group). (O–Q) RT and real-time PCR analysis of *Il6* expression in liver
 (O), eWAT (P), and spleen (Q) of control mice and mice at the indicated
 times after bilateral cast immobilization (n = 5-6 per group). (R) Serum
 concentration of TNF-a in control mice and mice at the indicated times
 after bilateral cast immobilization (n = 6 per group).
 **Figure_5_source_data_1.xlsx** **Description:**  **Figure 5:** (A) Change
 in rectal body temperature during cold exposure at 4°C for 4 h for WT (n =
 4 or 5) and IL-6 KO (n = 4 or 5) mice that had been subjected (or not) to
 bilateral cast immobilization for 24 h. (B and C) Concentrations of
 carbohydrate metabolites (B) and amino acids (C) in iBAT of IL-6 KO (–/–)
 and WT (+/+) mice without (control) or with bilateral cast immobilization
 for the indicated times. Results are presented as heat maps of the log~10~
 value of the fold change relative to control WT mice and are means of four
 mice in each group. (D–F) RT and real-time PCR analysis of the expression
 of SLC25A44 (D), BCKDHA (E), and CD36 (F) genes in iBAT of IL-6 KO and WT
 mice without (control) or with bilateral cast immobilization for 24 h (n =
 5 or 6). (G) Incorporation of [^3^H]leucine in iBAT of IL-6 KO  and WT
 mice without (control) or with bilateral cast immobilization for 24 h (n =
 4 or 5 per group). (H–J) BCAA (Val, Ile, and Leu, respectively)
 concentrations in soleus of IL-6 KO and WT mice without (control) or with
 bilateral cast immobilization for 1 or 7 days (n = 4 per group). **Figure
 5_figure supplement 1:** (A) RT and real-time PCR analysis of *Ucp1* mRNA
 abundance in iBAT of WT and IL-6 KO mice without (control) or with
 bilateral cast immobilization for 1 or 7 days (n = 5 or 6 per group). (B
 and C) Oxygen consumption rate for WT (B) and IL-6 KO (C) mice subjected
 (or not) to bilateral cast immobilization for 7 days (n = 4 per group).
 (D) Hematoxylin-eosin staining of iBAT from IL-6 KO and WT mice without
 (control) or with bilateral cast immobilization for 7 days. Scale bar, 50
 µm. (E and F) RER for WT (E) and IL-6 KO (F) mice subjected (or not) to
 bilateral cast immobilization for 7 days (n = 4 per group). (G) Weight of
 eWAT in WT (+/+) and IL-6 KO (-/- mice subjected (or not, control) to
 bilateral cast immobilization for 7 days (n = 5-7 per group). (H–J) Serum
 concentrations of BCAA (Val, Ile, and Leu, respectively) concentrations of
 IL-6 KO and WT mice without (control) or with bilateral cast
 immobilization for 1 or 7 days (n = 4 per group).
 **Figure_6_source_data_1.xlsx** **Description:**  **Figure 6:** (A–C)
 Concentrations of BCAAs (Val, Ile, and Leu, respectively) in the soleus of
 control IL-6 KO mice and at 3 h after intraperitoneal administration of
 recombinant IL-6 (rIL-6, 400 ng) or vehicle in IL-6 KO mice that had been
 subjected to bilateral cast immobilization for 24 h (n = 4 or 5 per
 group). (D) BCAA concentrations in C2C12 myotubes incubated in the absence
 or presence of rIL-6 (50 ng/ml) for 1 h (n = 6 independent experiments) .
 (E) Rectal core body temperature during cold exposure at 4°C for 1 h for
 control IL-6 KO mice and for IL-6 KO mice subjected to bilateral cast
 immobilization for 24 h and injected intraperitoneally with rIL-6 (400 ng)
 or vehicle 30 min before cold challenge (n = 5 per group). The white
 circles represent control mice, the red circles indicate cast-immobilized
 mice, and the red triangles represent cast-immobilized mice that were
 treated with recombinant IL-6 (rIL-6). (F) Rectal core body temperature
 during cold exposure at 4°C for 1 h for C57BL/6J mice with denervated iBAT
 subjected or not to bilateral cast immobilization and treatment with rIL-6
 or vehicle as in (E) (n = 4 per group). (G) Serum IL-6 concentration of
 mice subjected to surgical removal of iBAT and then subjected (or not,
 control) to bilateral cast immobilization for the indicated times (n = 4-6
 per group). The white circles represent control mice, the blue circles
 indicate C57BL/6J mice with denervated iBAT, and the blue triangles denote
 C57BL/6J mice with denervated iBAT that were treated with recombinant IL-6
 (rIL-6). (H) Weight of eWAT in mice subjected to surgical removal of iBAT
 (or to sham surgery) followed (or not, control) by bilateral cast
 immobilization for 7 days (n = 5-7 per group). (I–K) Concentrations of
 BCAAs (Val, Ile, and Leu, respectively) in soleus of mice subjected to
 surgical removal of iBAT and then subjected (or not, control) to bilateral
 cast immobilization for the indicated times (n = 4 per group). **Figure
 6_figure supplement 1:** (A) Amino acid concentrations in soleus of
 control IL-6 KO mice and at 3 h after intraperitoneal administration of
 recombinant IL-6 (rIL-6, 400 ng) or vehicle in IL-6 KO mice that had been
 subjected to bilateral cast immobilization for 24 h. Results are presented
 as heat maps of the log~10~ value of the fold change relative to control
 mice and are means of four or five mice in each group. (B and C) RT and
 real-time PCR analysis of *Saa3* (B) and *Socs3* (C) mRNA abundance in
 C2C12 myotubes incubated in the absence or presence of rIL-6 (50 ng/ml)
 for 1 h (n = 8 or 9 independent experiments). (D) Amounts of carbohydrate
 metabolites in iBAT of mice as in (A). Results are presented as heat maps
 of the log~10~ value of the fold change relative to control mice and are
 means of four mice in each group. (E) Amino acid concentrations in iBAT of
 mice as in (A). Results are presented as heat maps of the log~10~ value of
 the fold change relative to control mice and are means of four or five
 mice in each group. (F–H) RT and real-time PCR analysis of UCP1 (F),
 SLC25A44 (G), and BCKDHA (H) gene expression in iBAT of mice as in (A) (n
 = 5 per group). (I) Change in rectal core body temperature of mice of the
 indicated *Il6* genotypes during cold exposure at 4°C for 4 h (+/+ and
 –/–, n = 5; +/–, n = 6). (J) RT and real-time PCR analysis of *Ucp1* mRNA
 abundance in iBAT of mice of the indicated *Il6* genotypes (n = 5 or 6 per
 group). (K–M) RT and real-time PCR analysis of UCP1 (K), SLC25A44 (L), and
 BCKDHA (M) gene expression in cultured brown adipocytes incubated in the
 absence or presence of rIL-6 (50 ng/ml) or CL316 243 (1 µM) for 24 h (n =
 3 or 4 independent experiments). (N–P) RT and real-time PCR analysis of
 UCP1 (N), SLC25A44 (O) and BCKDHA (P) gene expression in iBAT of WT (+/+,
 n = 5 per group) or IL-6 KO (–/–, n = 8 per group) mice after
 intraperitoneal treatment with CL316 243 (0.1 mg/kg per day) or vehicle
 for 7 days. **Figure 6_figure supplement 2:** (A-E) RT and real-time PCR
 analysis of SLC43A1, SLC1A5, SLC7A5, BCAT2, and BCKDHA gene expression,
 respectively, in soleus of iBAT-depleted mice subjected (or not, control)
 to bilateral cast immobilization for the indicated times (n = 4–7 per
 group). (F) Change in soleus and gastrocnemius muscle weights of
 iBAT-depleted or sham-operated mice subjected to bilateral cast
 immobilization for 7 days (n = 6 per group).
 **Figure_7_source_data_1.xlsx** **Description:**  **Figure 7:** (A) RT and
 real-time PCR analysis of *Il6* expression in iBAT, soleus, liver, eWAT,
 and spleen of mice subjected (or not) to cold (4°C) exposure for 4 h
 (control, n = 6–9; cold exposure, n = 5 or 6). (B–D) Concentrations of
 BCAAs (Val, Ile, and Leu, respectively) in soleus of WT and IL-6 KO mice
 subjected (or not) to cold exposure at 4°C for 4 h (n = 4 per group). (E
 and F) RT and real-time PCR analysis of *Il6* expression in iBAT (E) and
 soleus (F) of control mice and mice subjected to restraint stress for 6 h
 (n = 4 per group). (G–I) Concentrations of BCAAs (Val, Ile, and Leu,
 respectively) in soleus of WT and IL-6 KO mice subjected (or not) to
 restraint stress for 6 h (n = 4 per group). **Figure 7_figure supplement
 1:** (A-E) RT and real-time PCR analysis of *Il6*, *Saa3*, *Socs3*,
 *Fbxo32* and *Trim63 *mRNA abundance in BBM of mice subjected (or not,
 control) to bilateral cast immobilization for the indicated times (n = 4
 per group). (F-H) BCAA (Val, Ile, and Leu, respectively) concentrations in
 BBM of mice subjected (or not, control) to bilateral cast immobilization
 for the indicated times (n = 4 per group). (I and J) RT and real-time PCR
 analysis of *Saa3* (I) and *Socs3* (J) mRNA abundance in soleus of mice
 subjected (or not) to cold (4°C) exposure for 4 h (control, n = 6; cold
 exposure, n = 5). (K and L) RT and real-time PCR analysis of *Fbox32* (K)
 and *Trim63* (L) mRNA abundance in soleus of mice subjected (or not) to
 cold (4°C) exposure for 4 h (control, n = 6; cold exposure, n = 5). (M and
 N) Weight of individual soleus (M) and gastrocnemius (N) muscles of
 control mice and mice subjected to. cold exposure at 4°C for 4 h (n = 5 or
 6 per group). (O) Amino acid concentrations in soleus of WT (+/+) and IL-6
 KO (–/–) mice subjected (or not) to cold exposure at 4°C for 4 h. Results
 are presented as heat maps of the log~10~ value of fold change relative to
 WT control mice and are means of four mice in each group. (P) BCAA
 concentrations in soleus of iBAT-depleted mice subjected (or not, control)
 to cold exposure at 4°C for 4 h (n = 3 or 4 per group). **Figure 7_figure
 supplement 2:** (A) RT and real-time PCR analysis of *Ucp1* mRNA abundance
 in iBAT of mice subjected (or not) to restraint stress for 6 h (n = 4 per
 group). (B and C) RT and real-time PCR analysis of *Saa3* (B) and *Socs3*
 (C) mRNA abundance in soleus of mice subjected (or not) to restraint
 stress for 6 h (n = 4 per group). (D-F) Weight of soleus (D),
 gastrocnemius (E) and EDL (F) in mice subjected to restraint stress for 6
 h (n = 4 per group). (G and H) RT and real-time PCR analysis of *Fbox32*
 (G) and *Trim63* (H) mRNA abundance in soleus of mice subjected (or not)
 to restraint stress for 6 h (n = 4 per group). (I) Amino acid
 concentrations in soleus of WT (+/+) and IL-6 KO (–/–) mice subjected (or
 not) to restraint stress for 6 h. Results are presented as heat maps of
 the log~10~ value of fold change relative to WT control mice and are means
 of four mice in each group.   ## Code/software Data files are provided in
 Excel format (.xlsx) and can be viewed with Microsoft Excel (version 2310)
 or free alternatives such as LibreOffice Calc. Statistical analyses were
 performed using JMP Pro (version 13, SAS Institute Inc.) and Statcel – The
 Useful Add-in Forms on Excel, 4th edition (OMS Publishing Inc.). These
 software packages were used for t tests, one-way and two-way ANOVA,
 Dunnett’s test, Tukey–Kramer test, and Bonferroni’s correction. No custom
 code or scripts were used. ## Access information This dataset is made
 publicly available for the first time via Dryad and is not archived
 elsewhere.